Itraconazole analogues and methods of use thereof

ABSTRACT

Disclosed herein are analogues of itraconazole that are both angiogenesis and hedgehog signaling pathway inhibitors. The compounds are expected to be useful in the treatment of cancer, particularly cancers that are dependent upon the hedgehog signaling pathway such as basal cell carcinoma and medulloblastoma.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of U.S. application Ser. No. 15/225,021filed on Aug. 1, 2016 which is a Continuation in Part ofPCT/US2015/013808 filed on Jan. 30, 2015, which claims priority to U.S.Provisional Application 61/934,714 filed on Feb. 1, 2104, which areincorporated herein by reference in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH & DEVELOPMENT

This invention was made with government support under 1R01CA190617-01awarded by the National Institutes of Health. The government has certainrights in the invention.

FIELD OF THE DISCLOSURE

The present disclosure is related to novel compounds having utility asanti-cancer agents, specifically novel derivatives of azole antifungals.

BACKGROUND

Identifying novel biological activities of FDA-approved drugs hasemerged as a viable strategy to expedite the drug discovery process. Thepharmacokinetic and toxicological profiles of these compounds arewell-understood and they are inherently “drug-like.” To this end, drugdevelopment researchers have made a concerted effort to incorporatesmall molecule libraries containing approved drugs in theirhigh-throughput screens. Recently, two such screens designed torepurpose FDA-approved compounds as anti-cancer chemotherapeuticsidentified the clinically efficacious antifungal itraconazole (ITZ) asboth an inhibitor of the hedgehog (Hh) signaling pathway (IC₅₀=690 nM)and angiogenesis (IC₅₀=160 nM

The Hh pathway is a developmental signaling pathway that plays a keyrole in directing growth and tissue patterning during embryonicdevelopment. Dysregulation of Hh signaling has been linked to thedevelopment of a variety of human tumors; most notably, basal cellcarcinoma (BCC) and medulloblastoma (MB). Recent years have seen thedevelopment of numerous small molecule Hh pathway inhibitors, themajority of which directly bind Smoothened (SMO), a 7-transmembraneGPCR-like receptor and key regulator of pathway signaling. The mostadvanced of these compounds, the small molecule GDC-0449(Vismodegib/Erivedge™), was approved by the FDA for the treatment ofmetastatic BCC, highlighting the clinical relevance of Hh pathwayinhibition. The importance of angiogenesis in tumor formation, growth,and metastasis is well-documented and numerous small molecules andbiologics that inhibit angiogenesis are clinically useful anti-canceragents.

While itraconazole has anti-cancer activity, it can have seriousdetrimental interactions with other commonly taken medications such asanticoagulants, statins and calcium channel blockers. It is thusdesirable to provide alternatives to itraconazole that maintain theanti-cancer properties, but that potentially do not have theside-effects observed for itraconazole.

BRIEF SUMMARY

In one aspect, included herein is a compound having the structure ofFormula (I)

-   -   wherein    -   Q is O or CH₂;    -   each Ar is independently unsubstituted or substituted aryl;    -   J is O or S;    -   R¹ is C₁₋₆alkyl optionally substituted with an amino, a C₁₋₆        alkylamino, a C₁₋₆ dialkylamino, an N-acylamino, —COOH, an aryl,        a heterocycle, pyrrolidine, pyrrole, or pyridinyl group;    -   R² is C₁₋₆ alkyl or unsubstituted or substituted aryl;    -   R³ is H or unsubstituted or substituted C₁₋₆ alkyl;    -   R⁴ is H or unsubstituted or substituted C₁₋₆ alkyl; or R³ and R⁴        join to form an unsubstituted or substituted 5- or 6-membered        ring with the —N—(=J)-N— moiety where R³ and    -   R⁴ form a unsubstituted or substituted C₂₋₃ carbohydryl group or        a unsubstituted or substituted C₁₋₂ carbohydryl group linked via        a nitrogen to a nitrogen of the —N—(=J)-N— moiety;    -   R⁵ is H, substituted or unsubstituted C₁₋₆ alkyl, C₁₋₆ alkoxy,        C₂₋₆ alkanoyl, C₁₋₆ alkoxcarbonyl, C₁₋₆ haloalkyl, wherein the        substituted C₁₋₆alkyl is substituted with 1, 2, or 3        substituents, each substituent is independently C₁₋₆ alkyl, —OH,        —COOH, cyano, nitro, C₁₋₆ monoalkylamine, C₁₋₆ dialkylamine,        C₁₋₆ haloalkyl, C₁₋₆ haloalkoxy;    -   a pharmaceutically acceptable salt, a stereoisomeric form        thereof, or a combination thereof.

In another aspect, included are pharmaceutical compositions includingthe disclosed compounds and a pharmaceutically acceptable excipient.

In yet another aspect, included herein are methods of treating cancer,particularly Hh-signaling pathway-dependent cancers, with the compoundsdisclosed herein, including posaconazole.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the structure and regions of itraconazole and Analogue 1.

FIG. 2 illustrates the Hh signaling pathway.

FIG. 3 shows the concentration-dependent down-regulation of endogenousGli1 mRNA in Hh-dependent mouse embryonic fibroblasts.

FIG. 4 shows the concentration-dependent down-regulation of endogenousGli1 mRNA in Hh-dependent BCC.

FIG. 5 shows the concentration-dependent inhibition of HUVECproliferation.

FIG. 6 shows the concentration-dependent inhibition of CYP3A4 byitraconazole and Analogue 1. These data are from a representativeexperiment performed in triplicate.

FIG. 7 shows Hh inhibitory activity of related azole antifungals. Valuesreported are percent inhibition of Hh signaling at 1 μM. Itraconazoleinhibition of Hh signaling at 1 μM=96%. Data from analogue 17a representpercent inhibition of Hh signaling in an Hh-dependent MB at the IC₉₀(IC₉₀ value not reported).

FIG. 8 shows the SAR/SPR generation protocol for itraconazole analogues.

FIG. 9 shows that Hh antagonists increase survival. (A) SHh driven humanMB xenografts infected with luciferase virus were transplanted into NSGmice. Bioluminescence images were taken prior to treatment (t0) andafter 36-day treatment with vehicle or LDE-225 (20 mg/kg daily). (B)Xenograft-bearing mice treated with vehicle or LDE-225 (20 mg/kg).

FIG. 10 shows tube formation assays for ITZ and its analogues. (A) DMSO,(B) Suramin, (C) ITZ, (D) 2a, (E) 18a, (F) 21a. All compounds wereevaluated at 10 μM and each image is a representative visual field froma single assay. Each assay was repeated at least three distinct times.

FIG. 11 shows a comparison of total tube length (A) and total tubejunctions (B) for ITZ, 2a, 18a, and 21a. DMSO (negative control) was setas 100% tube formation for analysis purposes. Suramin (10 μM) was usedas the positive control for each experiment and its ability to inhibittube length (51.9±7.8%) and tube junctions (57.6±6.1%) was consistent.Data represent the Ave ±SEM of at least 3 separate experiments in which≧5 fields of vision were quantified using ImageJ software.

The above-described and other features will be appreciated andunderstood by those skilled in the art from the following detaileddescription, drawings, and appended claims.

DETAILED DESCRIPTION

Disclosed herein are itraconazole (ITZ) and posaconazole (PSZ) analogueswith novel structures that were designed based on systematic explorationof the ITZ and PSZ scaffolds to identify structural modifications thatenhance inhibition of Hh signaling and maintain this activity againstSMO mutants that confer resistance against vismodegib or other Hhinhibitors. The ITZ and PSZ analogues are angiogenesis inhibitors andare thus particularly useful as anti-cancer agents, for example, totreat cancers that are dependent upon the Hh signaling pathway. Inanother aspect, the ITZ and PSZ analogs are useful to treat cancers thatare resistant to Vismodegib. In another embodiment, PSZ is disclosed asuseful to treat cancer, including for example, Hh signalingpathway-dependent cancers.

The anti-fungal activity of ITZ is a result of its inhibition oflanosterol 14α-demethylase (14LDM/CYP51), a cytochrome P450 enzyme thatplays a crucial role in the biosynthesis of ergosterol, which is a majorcomponent of fungal cell membranes. The N4 of the ITZ triazole moietyinteracts with the heme group in 14LDM, preventing coordination of themolecular oxygen required to initiate oxidation. This ability tocoordinate the heme of CYP450s is also responsible for its most commondetrimental side effect, inhibition of CYP3A4. CYP3A4 inhibition resultsin multiple drug-drug interactions and contraindications and requirescareful monitoring of diet and drug regimens when ITZ is administered.In addition to the triazole containing “left-hand” portion of thescaffold, ITZ also contains a central phenyl-piperizine-phenyl linkerregion and a “right-side” triazolone/side chain region (FIG. 1). Withinthe context of its anti-fungal activity, the extended linker andtriazolone/side chain regions are not essential for binding the activesite of CYP51; however, they do interact with several amino acidresidues in the substrate access channel. ITZ contains three chiralcenters (2, 4, and 2′), and can potentially exist as a mixture of eightdistinct stereoisomers; however, formation of the cis-configurationsaround the dioxolane ring predominates during its synthesis andpharmaceutical preparations of ITZ are typically administered as a1:1:1:1 mixture of the cis racemates.

In one aspect, described herein are ITZ analogues in which the triazolemoiety has been removed such as Analogue 1. FIG. 1 compares thestructures of ITZ and Analogue 1. Analogue 1, for example, retains itsHh inhibitory activity and does not affect CYP3A4. In addition, alsoincluded herein are ITZ analogues in which the triazole ring has beenreplaced with various isosteres. Thus, a novel change to the ITZscaffold has been identified that improves activity and reducesdetrimental side effects. Additional analogues of ITZ and PSZ are alsodescribed herein.

Specifically, described herein are ITZ and PSZ analogues, wherein theITZ and PSZ analogues are lacking the triazole moiety, or wherein thetriazole moiety has been modified. In certain aspects, the ITZ and PSZanalogues are effective inhibitors of the Hh pathway, do not inhibitCYP3A4, and/or have anti-angiogenic activity. Overall, the strategy forthe design of the ITZ and PSZ analogues was to make conservative andsystematic modifications to separate regions of the ITZ and PSZscaffolds. Because removal of the triazole functionality abrogates theoff-target CYP3A4 inhibition and does not affect Hh inhibition, theanalogues will not incorporate the triazole functionality.

The ITZ and PSZ analogues are compounds having the structure of Formula(I)

-   -   wherein    -   Q is O or CH₂, specifically O;    -   each Ar is independently unsubstituted or substituted aryl,        specifically phenyl, pyridine, pyrazine, or pyridazine, and more        specifically phenyl;    -   J is O or S, specifically O;    -   R¹ is C₁₋₆ alkyl optionally substituted with an amino, a C₁₋₆        alkylamino, a C₁₋₆ dialkylamino, an N-acylamino, —COOH, an aryl,        a heterocycle, pyrrolidine, pyrrole, or pyridinyl group,        specifically R¹ is methyl, optionally substituted with        1-pyrrole, 3-pyridine, 4-pyridine, phenyl, m-aminophenyl,        p-aminophenyl, acetylamine, 1-pyrrolidine, amino, or        dimethylamino, and more specifically R¹ is methyl;    -   R² is C₁₋₆ alkyl or unsubstituted or substituted aryl,        specifically unsubstituted or substituted phenyl, and more        specifically R² is 2,4-dichlorophenyl or 2,4-difluorophenyl;    -   R³ is H or unsubstituted or substituted C₁₋₆ alkyl;    -   R⁴ is H or unsubstituted or substituted C₁₋₆ alkyl; or R³ and R⁴        join to form an unsubstituted or substituted 5- or 6-membered        ring with the —N—(=J)-N— moiety where R³ and    -   R⁴ form a unsubstituted or substituted C₂₋₃ carbohydryl group or        a unsubstituted or substituted C₁₋₂ carbohydryl group linked via        a nitrogen to a nitrogen of the —N—(=J)-N— moiety;    -   R⁵ is H, substituted or unsubstituted C₁₋₆ alkyl, C₁₋₆ alkoxy,        C₂₋₆ alkanoyl, C₁₋₆ alkoxcarbonyl, C₁₋₆ haloalkyl, wherein the        substituted C₁₋₆ alkyl is substituted with 1, 2, or 3        substituents, each substituent is independently C₁₋₆ alkyl, —OH,        —COOH, cyano, nitro, C₁₋₆ monoalkylamine, C₁₋₆ dialkylamine,        C₁₋₆ haloalkyl, C₁₋₆ haloalkoxy;    -   a pharmaceutically acceptable salt, a stereoisomeric form        thereof, or a combination thereof.

Specifically, in certain embodiments, the compounds of Formula (I)exclude itraconazole (CAS registry no. 84625-61-6) and posaconazole (CASregistry no. 171228-49-2).

In an embodiment, R³ and R⁴ join to form an unsubstituted or substituted5- or 6-membered ring with the —N—(=J)-N— moiety where R³ and R⁴ form aunsubstituted or substituted C₂₋₃ carbohydryl group such as —CH═CH—,—CH₂—CH₂—, —CH₂—CH₂—CH₂—, or a unsubstituted or substituted C₁₋₂carbohydryl group linked via a nitrogen to a nitrogen of the —N—(=J)-N—moiety, e.g. —CH═N—.

In an embodiment, R⁵ is propyl; 2′-sec-butyl, the R isomer, the Sisomer, a racemate or any enantiomerically enriched form;2-hydroxypentan-3-yl, the 2R,3R-isomer, the 2S,3S, isomer, the 2R,3S,isomer, the 2S,3R isomer, or any diastereomerically enriched form;2-hydroxyprop-2-yl; or 2-hydroxyprop-1-yl, the R isomer, the S isomer, aracemate, or any enantiomerically enriched form.

Within Formula (I), the compounds can be prepared in racemic form, orany optically enriched form. Exemplary stereoisomers of Formula (I)include Formulae (I-1), (I-2), (I-3), or (I-4):

In another embodiment, the compounds have the structure of Formula (Ia)

-   -   wherein    -   Q, J, R¹, R², R³, R⁴, and R⁵ are as previously defined, and        wherein    -   each one of X¹, X², Y¹, Y², Z¹, and Z² independently is CH,        CCH₃, or N.

Specifically, in certain embodiments, Formula (Ia) excludes itraconazole(CAS registry no. 84625-61-6) and posaconazole (CAS registry no.171228-49-2). In several embodiments, a compound according to Formula(Ia) is included, wherein

-   -   X¹ is N and Y¹, Z¹, X², Y², and Z² are CH;    -   Y¹ is N and X¹, Z¹, X², Y², and Z² are CH;    -   X¹ and Y¹ are N and Z¹, X², Y², and Z² are CH;    -   X¹ and Z¹ are N and Y¹, X², Y², and Z² are CH;    -   X² is N and X¹, Y¹, Z¹, Y², and Z² are CH;    -   Y² is N and X¹, Y¹, Z¹, X², and Z² are CH;    -   X² and Y² are N and X¹, Y¹, Z¹, and Z² are CH; or    -   X² and Z² are N and X¹, Y¹, Z¹, and Y² are CH.

It will be understood that the corresponding chiral Formulae (Ia-1),(Ia-2), (Ia-3), and (Ia-4) are included.

In another embodiment, the compounds have the structure of Formula (Ib)

-   -   wherein    -   Q and R⁵ are as previously defined, and wherein    -   R⁶ and R⁷ are each independently H, halo, C₁₋₆ alkyl, C₁₋₆        alkoxy, C₂₋₆ alkanoyl, C₁₋₆ alkoxcarbonyl, —NH₂, —OH, —COOH,        cyano, nitro, C₁₋₆ monoalkylamine, C₁₋₆ dialkylamine, C₁₋₆        haloalkyl, or C₁₋₆ haloalkoxy, specifically halo, and more        specifically R⁶ and R⁷ are each independently Cl or F.

Specifically, in certain embodiments, Formula (Ib) excludes itraconazole(CAS registry no. 84625-61-6) and posaconazole (CAS registry no.171228-49-2).

It will be understood that the corresponding chiral Formulae (Ib-1),(Ib-2), (Ib-3), and (Ib-4) are included.

In certain situations, the compounds of Formulae I, Ia and Ib maycontain one or more asymmetric elements such as stereogenic centers,stereogenic axes and the like, e.g., asymmetric carbon atoms, so thatthe compounds can exist in different stereoisomeric forms. Thesecompounds can be, for example, racemates or optically active forms. Forcompounds with two or more asymmetric elements, these compounds canadditionally be mixtures of diastereomers. For compounds havingasymmetric centers, it should be understood that all of the opticalisomers and mixtures thereof are encompassed. In addition, compoundswith double bonds may occur in Z- and E-forms, with all isomeric formsof the compounds being included in the present disclosure. In thesesituations, the single enantiomers, i.e., optically active forms, can beobtained by asymmetric synthesis, synthesis from optically pureprecursors, or by resolution of the racemates. Resolution of theracemates can also be accomplished, for example, by conventional methodssuch as crystallization in the presence of a resolving agent, orchromatography, using, for example a chiral HPLC column.

The term “substituted”, as used herein, means that any one or morehydrogens on the designated atom or group is replaced with a selectionfrom the indicated group, provided that the designated atom's normalvalence is not exceeded. When a substituent is oxo (i.e., ═O), then 2hydrogens on the atom are replaced. When aromatic moieties aresubstituted by an oxo group, the aromatic ring is replaced by thecorresponding partially unsaturated ring. For example a pyridyl groupsubstituted by oxo is a pyridone. Combinations of substituents and/orvariables are permissible only if such combinations result in stablecompounds or useful synthetic intermediates. A stable compound or stablestructure is meant to imply a compound that is sufficiently robust tosurvive isolation from a reaction mixture, and subsequent formulationinto an effective therapeutic agent.

A dash (“-”) that is not between two letters or symbols is used toindicate a point of attachment for a substituent. For example, —COOH isattached through the carbon atom.

As used herein, “alkyl” is intended to include both branched andstraight-chain saturated aliphatic hydrocarbon groups, having thespecified number of carbon atoms. Thus, the term C₁-C₆ alkyl as usedherein includes alkyl groups having from 1 to about 6 carbon atoms. WhenC₀-C_(n) alkyl is used herein in conjunction with another group, forexample, phenylC₀-C₄ alkyl, the indicated group, in this case phenyl, iseither directly bound by a single covalent bond (C₀), or attached by analkyl chain having the specified number of carbon atoms, in this casefrom 1 to about 2 carbon atoms. Examples of alkyl include, but are notlimited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl,n-pentyl, and sec-pentyl.

“Alkenyl” as used herein, indicates hydrocarbon chains of either astraight or branched configuration comprising one or more unsaturatedcarbon-carbon bonds, which may occur in any stable point along thechain, such as ethenyl and propenyl.

“Alkynyl” as used herein, indicates hydrocarbon chains of either astraight or branched configuration comprising one or more triplecarbon-carbon bonds that may occur in any stable point along the chain,such as ethynyl and propynyl.

“Alkoxy” represents an alkyl group as defined above with the indicatednumber of carbon atoms attached through an oxygen bridge. Examples ofalkoxy include, but are not limited to, methoxy, ethoxy, n-propoxy,propoxy, n-butoxy, 2-butoxy, t-butoxy, n-pentoxy, 2-pentoxy, 3-pentoxy,isopentoxy, neopentoxy, n-hexoxy, 2-hexoxy, 3-hexoxy, and3-methylpentoxy.

“Alkanoyl” indicates an alkyl group as defined above, attached through aketo (—(C═O)—) bridge. Alkanoyl groups have the indicated number ofcarbon atoms, with the carbon of the keto group being included in thenumbered carbon atoms. For example a C₂alkanoyl group is an acetyl grouphaving the formula CH₃(C═O)—.

The term “alkoxycarbonyl” indicates an alkoxy group, as defined above,having the indicated number of carbon atoms, attached through a ketolinkage. The carbon of the keto linker is not included in the numbering,thus a C₂alkoxycarbonyl has the formula CH₃CH₂O(C═O)—.

The term “alkylcarboxamide” indicates an alkyl group, as defined above,having the indicated number of carbon atoms, attached through acarboxamide linkage, i.e., a —CONH₂ linkage, where one or both of theamino hydrogens is replaced by an alkyl group. Alkylcarboxamide groupsmay be mono- or di-alkylcarboxamide groups, such an ethylcarboxamide ordimethylcarboxamide.

As used herein, the term “mono- or di-alkylamino” indicates secondary ortertiary alkyl amino groups, wherein the alkyl groups are as definedabove and have the indicated number of carbon atoms. The point ofattachment of the alkylamino group is on the nitrogen. Examples of mono-and di-alkylamino groups include ethylamino, dimethylamino, andmethyl-propyl-amino.

As used herein, the term “aryl” indicates aromatic groups containingonly carbon in the aromatic ring or rings. Such aromatic groups may befurther substituted with carbon or non-carbon atoms or groups. Typicalaryl groups contain 1 to 3 separate, fused, or pendant rings and from 6to about 18 ring atoms, without heteroatoms as ring members. Whereindicated aryl groups may be substituted. Such substitution may includefusion to a 5 to 7-membered saturated cyclic group that optionallycontains 1 or 2 heteroatoms independently chosen from N, O, and S, toform, for example, a 3,4-methylenedioxy-phenyl group. Aryl groupsinclude, for example, phenyl, naphthyl, including 1-naphthyl and2-naphthyl, and bi-phenyl.

In the term “(aryl)alkyl”, aryl and alkyl are as defined above, and thepoint of attachment is on the alkyl group. This term encompasses, but isnot limited to, benzyl, phenylethyl, and piperonyl. Likewise, in theterm (aryl)carbohydryl, aryl and carbohydryl are as defined above andthe point of attachment is on the carbohydryl group, for example aphenylpropen-1-yl group.

“Carbohydryl” as used herein, includes both branched and straight-chainhydrocarbon groups, which are saturated or unsaturated, having thespecified number of carbon atoms.

“Cycloalkyl” as used herein, indicates saturated hydrocarbon ringgroups, having the specified number of carbon atoms, usually from 3 toabout 8 ring carbon atoms, or from 3 to about 7 carbon atoms. Examplesof cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, orcyclohexyl as well as bridged or caged saturated ring groups such asnorbornane or adamantane.

“Haloalkyl” indicates both branched and straight-chain saturatedaliphatic hydrocarbon groups having the specified number of carbonatoms, substituted with 1 or more halogen atoms, generally up to themaximum allowable number of halogen atoms. Examples of haloalkylinclude, but are not limited to, trifluoromethyl, difluoromethyl,2-fluoroethyl, and penta-fluoroethyl.

“Haloalkoxy” indicates a haloalkyl group as defined above attachedthrough an oxygen bridge.

“Halo” or “halogen” as used herein refers to fluoro, chloro, bromo, oriodo.

As used herein, “heteroaryl” indicates a stable 5- to 7-memberedmonocyclic or 7-to 10-membered bicyclic heterocyclic ring which containsat least 1 aromatic ring that contains from 1 to 4, or preferably from 1to 3, heteroatoms chosen from N, O, and S, with remaining ring atomsbeing carbon. When the total number of S and O atoms in the heteroarylgroup exceeds 1, these heteroatoms are not adjacent to one another. Itis preferred that the total number of S and O atoms in the heteroarylgroup is not more than 2. Examples of heteroaryl groups include, but arenot limited to, pyridyl, indolyl, pyrimidinyl, pyridizinyl, pyrazinyl,imidazolyl, oxazolyl, furanyl, thiophenyl, thiazolyl, triazolyl,tetrazolyl, isoxazolyl, quinolinyl, pyrrolyl, pyrazolyl, and5,6,7,8-tetrahydroisoquinoline. In the term “heteroarylalkyl,”heteroaryl and alkyl are as defined above, and the point of attachmentis on the alkyl group. This term encompasses, but is not limited to,pyridylmethyl, thiophenylmethyl, and pyrrolyl(1-ethyl).

The term “heterocycloalkyl” is used to indicate saturated cyclic groupscontaining from 1 to about 3 heteroatoms chosen from N, O, and S, withremaining ring atoms being carbon. Heterocycloalkyl groups have from 3to about 8 ring atoms, and more typically have from 5 to 7 ring atoms. AC₂-C₇heterocycloalkyl group contains from 2 to about 7 carbon ring atomsand at least one ring atom chosen from N, O, and S. Examples ofheterocycloalkyl groups include morpholinyl, piperazinyl, piperidinyl,and pyrrolidinyl groups.

“Pharmaceutically acceptable salts” includes derivatives of thedisclosed compounds wherein the parent compound is modified by making anacid or base salt thereof, and further refers to pharmaceuticallyacceptable solvates of such compounds and such salts. Examples ofpharmaceutically acceptable salts include, but are not limited to,mineral or organic acid salts of basic residues such as amines; alkalior organic salts of acidic residues such as carboxylic acids; and thelike. The pharmaceutically acceptable salts include the conventionalsalts and the quaternary ammonium salts of the parent compound formed,for example, from inorganic or organic acids. For example, conventionalacid salts include those derived from inorganic acids such ashydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric andthe like; and the salts prepared from organic acids such as acetic,propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric,ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic,benzoic, salicylic, mesylic, esylic, besylic, sulfanilic,2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethanedisulfonic, oxalic, isethionic, HOOC—(CH₂)_(n)—COOH where n is 0-4, andthe like. The pharmaceutically acceptable salts of the present inventioncan be synthesized from a parent compound that contains a basic oracidic moiety by conventional chemical methods. Generally, such saltscan be prepared by reacting free acid forms of these compounds with astoichiometric amount of the appropriate base (such as Na, Ca, Mg, or Khydroxide, carbonate, bicarbonate, or the like), or by reacting freebase forms of these compounds with a stoichiometric amount of theappropriate acid. Such reactions are typically carried out in water orin an organic solvent, or in a mixture of the two. Generally,non-aqueous media like ether, ethyl acetate, ethanol, isopropanol, oracetonitrile are preferred, where practicable.

The ITZ and PSZ analogues disclosed herein as well as PSZ are expectedto have activity as angiogenesis inhibitors and thus to be useful in thetreatment of cancer. In certain embodiments, the compounds disclosedherein and PSZ are inhibitors of the Hh signaling pathway and to beparticularly useful in the treatment of cancers that are dependent uponthe Hh signaling pathway. In cells regulated by the Hh pathway, signaltransmission is controlled through a cascade that determines the balancebetween activator and repressor forms of the Gli family of transcriptionfactors (FIG. 2). In the absence of Hh ligand, Patched (Ptch) suppressesthe activity of Smoothened (Smo), a seven-transmembrane protein that isnormally observed in endosomes. This inhibition ultimately results inthe generation of N-terminal truncated Gli proteins, Gli^(R), that actas repressors of Hh-responsive genes. Binding of an Hh ligand to Ptchabolishes its inhibition of Smo, leading to the production offull-length Gli activator (Gli^(A)) proteins and resulting in expressionof Hh target genes that control proper cell fate determination.Dysregulation of the pathway causes constitutive activation, resultingin uncontrolled proliferation and tumor growth; most notably, in basalcell carcinoma (BCC) and MB. Other cancers that may be treated with Hhsignaling pathway inhibitors include chronic myeloid leukemia, lungcancer, prostate cancer, pancreatic cancer and bone cancer.

BCC is the most commonly diagnosed form of cancer in persons of Europeanancestry (affecting approximately 1 million Americans annually). It hasbeen estimated that approximately 30% of Caucasians living in areas ofhigh sun exposure will develop a BCC during their lifetime and theincidence of BCC in younger populations (especially young females) isrising. While BCC is rarely fatal, it can result in significantmorbidity and the large number of affected individuals presents anincreasing health burden.

MB is the most common malignant central nervous system tumor inchildren, accounting for approximately 20% of pediatric brain tumors andmost commonly occurring in children under the age of 8 (40% before theage of 5). Current therapy for pediatric MB patients includes surgeryfollowed by radiation and high-dose chemotherapy. While survival ratesfor pediatric MB patients have improved over the last ten years,long-term side effects of this course of treatment can includeneurocognitive and endocrine deficits as well as growth impairment. Inaddition, these patients are at an increased risk of developingsecondary tumors later in life. Uncontrolled activation of Hh signaling,including both mutation and amplification of key pathway components, hasbeen implicated in approximately 25% of MBs.

In certain embodiments, the compounds described herein are administeredto a patient or subject. A “patient” or “subject”, used equivalentlyherein, means mammals and non-mammals. “Mammals” means a member of theclass Mammalia including, but not limited to, humans, non-human primatessuch as chimpanzees and other apes and monkey species; farm animals suchas cattle, horses, sheep, goats, and swine; domestic animals such asrabbits, dogs, and cats; laboratory animals including rodents, such asrats, mice, and guinea pigs; and the like. Examples of non-mammalsinclude, but are not limited to, birds, and the like. The term “subject”does not denote a particular age or sex.

The phrase “effective amount,” as used herein, means an amount of anagent which is sufficient enough to significantly and positively modifysymptoms and/or conditions to be treated (e.g., provide a positiveclinical response). The effective amount of an active ingredient for usein a pharmaceutical composition will vary with the particular conditionbeing treated, the severity of the condition, the duration of thetreatment, the nature of concurrent therapy, the particular activeingredient(s) being employed, the particular pharmaceutically-acceptableexcipient(s)/carrier(s) utilized, and like factors within the knowledgeand expertise of the attending physician. In general, the use of theminimum dosage that is sufficient to provide effective therapy ispreferred. Patients may generally be monitored for therapeuticeffectiveness using assays suitable for the condition being treated orprevented, which will be familiar to those of ordinary skill in the art.

The amount of compound effective for any indicated condition will, ofcourse, vary with the individual subject being treated and is ultimatelyat the discretion of the medical or veterinary practitioner. The factorsto be considered include the condition being treated, the route ofadministration, the nature of the formulation, the subject's bodyweight, surface area, age and general condition, and the particularcompound to be administered. In general, a suitable effective dose is inthe range of about 0.1 to about 500 mg/kg body weight per day,preferably in the range of about 5 to about 350 mg/kg per day. The totaldaily dose may be given as a single dose, multiple doses, e. g., two tosix times per day, or by intravenous infusion for a selected duration.Dosages above or below the range cited above may be administered to theindividual patient if desired and necessary.

As used herein, “pharmaceutical composition” means therapeuticallyeffective amounts of the compound together with a pharmaceuticallyacceptable excipient, such as diluents, preservatives, solubilizers,emulsifiers, and adjuvants. As used herein “pharmaceutically acceptableexcipients” are well known to those skilled in the art.

Tablets and capsules for oral administration may be in unit dose form,and may contain conventional excipients such as binding agents, forexample syrup, acacia, gelatin, sorbitol, tragacanth, orpolyvinyl-pyrrolidone; fillers for example lactose, sugar, maize-starch,calcium phosphate, sorbitol or glycine; tabletting lubricant, forexample magnesium stearate, talc, polyethylene glycol or silica;disintegrants for example potato starch, or acceptable wetting agentssuch as sodium lauryl sulphate. The tablets may be coated according tomethods well known in normal pharmaceutical practice. Oral liquidpreparations may be in the form of, for example, aqueous or oilysuspensions, solutions, emulsions, syrups or elixirs, or may bepresented as a dry product for reconstitution with water or othersuitable vehicle before use. Such liquid preparations may containconventional additives such as suspending agents, for example sorbitol,syrup, methyl cellulose, glucose syrup, gelatin hydrogenated ediblefats; emulsifying agents, for example lecithin, sorbitan monooleate, oracacia; non-aqueous vehicles (which may include edible oils), forexample almond oil, fractionated coconut oil, oily esters such asglycerine, propylene glycol, or ethyl alcohol; preservatives, forexample methyl or propyl p-hydroxybenzoate or sorbic acid, and ifdesired conventional flavoring or coloring agents.

For topical application to the skin, the drug may be made up into acream, lotion or ointment. Cream or ointment formulations which may beused for the drug are conventional formulations well known in the art.Topical administration includes transdermal formulations such aspatches.

For topical application to the eye, the compounds may be made up into asolution or suspension in a suitable sterile aqueous or non aqueousvehicle. Additives, for instance buffers such as sodium metabisulphiteor disodium edeate; preservatives including bactericidal and fungicidalagents such as phenyl mercuric acetate or nitrate, benzalkonium chlorideor chlorhexidine, and thickening agents such as hypromellose may also beincluded.

The active ingredient may also be administered parenterally in a sterilemedium, either subcutaneously, or intravenously, or intramuscularly, orintrasternally, or by infusion techniques, in the form of sterileinjectable aqueous or oleaginous suspensions. Depending on the vehicleand concentration used, the drug can either be suspended or dissolved inthe vehicle. Advantageously, adjuvants such as a local anaesthetic,preservative and buffering agents can be dissolved in the vehicle.

Pharmaceutical compositions may conveniently be presented in unit dosageform and may be prepared by any of the methods well known in the art ofpharmacy. The term “unit dosage” or “unit dose” means a predeterminedamount of the active ingredient sufficient to be effective for treatingan indicated activity or condition. Making each type of pharmaceuticalcomposition includes the step of bringing the active compound intoassociation with a carrier and one or more optional accessoryingredients. In general, the formulations are prepared by uniformly andintimately bringing the active compound into association with a liquidor solid carrier and then, if necessary, shaping the product into thedesired unit dosage form.

The invention is further illustrated by the following non-limitingexamples.

Examples Example 1: Preparation of Analogue 1

The preparation of Analogue 1 predominantly utilized syntheticprocedures that have been previously disclosed for the synthesis of ITZand related azole anti-fungals. Ketalization of2′,4′-dichloroacetophenone with glycerol in the presence ofpara-toluenesulfonic acid provided dioxolane 3; the primary hydroxyl ofwhich was subsequently tosylated to afford key des-triazole intermediate4 (Scheme 1).

^(a)Reagents and conditions: (a) p-TsOH (0.016 eq), reflux, 24 h, 88%;(b) p-TsCl, TEA, DCM, 85%.

Reduction of the nitro functionality in 5 via palladium-catalyzedtransfer hydrogenation with hydrazine monohydrate provided the aniline,which was converted to triazolone 9 through the well-characterizedphenylcarbamate and semicarbazide intermediates (Scheme 2). Alkylationof the triazolone amine with 2-bromobutane followed by removal of themethyl moiety afforded phenol 11. Base-catalyzed displacement of thetosyl moiety in 4 by the phenol provided the final des-triazole Analogue1.

^(a)Reagents and conditions: (a) Pd/C, NH₂NH₂—H₂O (10 eq), reflux, 3.5h, 71%; (b) Pyr (17 eq), ClCOOPh (1.1 eq), 3 h, 90%; (c) NH₂NH₂—H₂O (5.5eq), reflux, 3 h, quant; (d) formamidine acetate (4.5 eq), reflux, 3 h,86%; (e) 2-bromobutane (3.0 eq), 18-crown-6 (1.2 eq), 12 h, 67%; (f)HBr, overnight, 130° C., 83%; (g) intermediate 4 (0.9 eq), NaH (4.5 eq),DMSO, 50-90° C., 12 h, 34%.

Example 2: In Vitro Inhibition of Hh Signaling by Analogue 1

To determine whether the triazole functionality was required forinhibition of Hh signaling, the ability of Analogue 1 to down-regulatethe well-characterized Hh pathway target gene Gli1 in the Hh-dependentC3H10T1/2 cell line was determined. In this cellular model, activationof the pathway with exogenous recombinant Hh ligand results in acharacteristic up-regulation of Gli1 mRNA, while concomitantadministration of a pathway inhibitor reduces Gli1 expression. Analogue1 maintained potent down-regulation of Gli1 in this cell line with anIC₅₀ value comparable to ITZ (140 and 74 nM, respectively), clearlydemonstrating that the triazole functionality is not required for Hhinhibition (FIG. 3 and Table 1). In addition, both ITZ and Analogue 1were evaluated for their ability to down-regulate endogenous Gli1expression in ASZ cells, a murine BCC cell line that has been utilizedpreviously by our lab and others as an early stage in vitro model ofHh-dependent cancer. Both ITZ and Analogue 1 reduced Gli1 expression ina concentration-dependent fashion with IC₅₀ values comparable to thosedetermined in the C3H10T1/2 cell line (FIG. 4).

TABLE 1 Anti-Hh and anti-proliferative activity of ITZ and Analogue 1.HUVEC Hh pathway inhibition^(a) Anti- Analogue C3H10T1/2 ASZproliferation GDC-0449  0.082 ± 0.02^(b) 0.040 ± 0.01  — ITZ 0.074 ±0.02 0.14 ± 0.02 0.45 ± 0.14 Analogue 1  0.14 ± 0.04 0.17 ± 0.04 23.8 ±6.7  ^(a)Measured as down-regulation of endogenous Gli1 mRNA. ^(b)Valuesare in μM and represent Mean ± SEM for at least three separateexperiments performed in triplicate.

Example 3: Anti-Angiogenic Activity of Analogue 1

To assess the anti-angiogenic activity of Analogue 1 compared to ITZ,their ability to inhibit proliferation in human umbilical veinepithelial cells (HUVECs) was evaluated. In vivo angiogenesis isdependent on endothelial cell proliferation; therefore, the inhibitionof HUVEC proliferation is commonly utilized as an early stage in vitromodel of anti-angiogenic activity. In addition, identification of ITZ asan anti-angiogenic compound was established through its ability toinhibit the in vitro proliferation of HUVECs. Interestingly, while ITZdemonstrated potent inhibition of HUVEC proliferation at levelscomparable to those previously reported (GI₅₀=0.45 μM), Analogue 1 wassignificantly less active in this assay (GI₅₀=23.8 μM) (FIG. 5). Tofurther explore the anti-angiogenic properties of both ITZ and Analogue1, their ability to inhibit tube formation in HUVECs grown on Matrigelwas evaluated. Under normal conditions, HUVECs plated and grown onMatrigel migrate towards each other, align, and form tubes that resemblein vivo capillary beds. This assay is generally considered a more robustmodel of angiogenesis as it requires several aspects of proper vesselformation, including adhesion, migration, and tube formation. Both ITZand Analogue 1 significantly inhibited tube formation in this model at0.1 and 1 μM (FIG. 10, 11); however, only Analogue 1 demonstratedsignificant inhibition at 10 nM. Neither compound demonstratedsignificant anti-proliferative activity at these concentrations at the24 hour time point utilized for the tube formation assay (data notshown). Taken together, these results indicate that removal of thetriazole does not affect the anti-angiogenic activity of the ITZscaffold while also suggesting an additional mechanism of generaltoxicity for ITZ in endothelial cells.

Example 4: Effect of Analogue 1 on CYP3A4 Activity

As noted above, a major side-effect of ITZ and other members of theazole class of anti-fungals is potent inhibition of the key metabolicenzyme Cyp3A4. As expected, removal of the triazole moiety completelyabolished the anti-CYP3A4 activity of the scaffold. While ITZsignificantly inhibited CYP3A4 activity, Analogue 1 was completelyinactive (IC₅₀ values 50.4 nM and >10 μM, respectively, FIG. 8).

Discussion of Validation of Analogue 1

Analogue 1 is an improved lead compound based on the ITZ scaffold fordevelopment as an anti-cancer agent. While removal of the triazolefunctionality had no effect on the ability of Analogue 1 to inhibit Hhsignaling or angiogenesis, it completely abolished its inhibitoryeffects on CYP3A4. This is of particular importance for cancer patientswho are routinely administered drug regimens that may contain multipledrugs whose circulating concentrations are affected by inhibition ofCYP3A4. In addition, the results verify that the known target of ITZ,14LDM, is not responsible for its intriguing anti-cancer properties.

Example 5: Ability of Related Antifungals to Inhibit the PathwaySignaling in C3H10T1/2 Cells at 1 μM

In addition to Analogue 1, a series of related azole anti-fungals havebeen studied for their ability to inhibit pathway signaling in C3H10T1/2cells at 1 μM. The results from these studies have informed the designand preparation of the analogues described herein (FIG. 9). First, theonly compound that demonstrated Hh inhibition comparable to ITZ wasPosaconazole (PSZ, % Hh inhibition=96% and 95%, respectively). PSZ astructurally-related, FDA approved triazole antifungal that containseach general structural region of ITZ (triazole, linker, triazolone, andside chain). Second, the structural differences between the triazoleregions of ITZ and PSZ suggest that this region is amenable tomodification beyond truncation to remove the triazole ring. Third, whileremoval of the side chain of ITZ had minimal effects on Hh inhibition(analogue 17a), removal of the phenyl/triazolone/side chain region ofthe scaffold, as in terconazole and ketoconazole, results in asignificant reduction in Hh inhibitory activity, suggesting thesemoieties are required for potent Hh inhibition. Further analysisdemonstrated that PSZ inhibited Hh signaling in aconcentration-dependent fashion similar to ITZ in C3H10T1/2 cells(IC₅₀=10 nM).

Example 6: Preparation of a Des-Triazole PSZ Analogue

A des-triazole PSZ analogue (39, Scheme 5) that is designed to provideadditional SAR and stability information for the ITZ/PSZ scaffold willbe prepared. First, this analogue will provide evidence as to whetherthe triazole functionality is non-essential for the anti-Hh activity ofthe PSZ scaffold. Second, in combination with the data from thecis-2R,4R dioxolane ITZ analogues (25, 29, and 33), this analogue willhelp determine the optimal orientation around thedioxolane/tetrahydrofuran (THF) moieties in the triazole region.Finally, oxidation at C-5 of the dioxolane moiety in ITZ and subsequentring opening was shown to be a major metabolic fate of the parentcompound. By contrast, significant metabolism of the THF moiety in PSZhas not been identified; therefore, the evaluation of 39 will provideimportant stability comparisons between the dioxolane and THF moietiesof the des-triazole analogues.

Carboxylic acid 35 is readily available in two synthetic steps from1,3-difluorobenzene and succinic anhydride. Activation of 35 withpivaloyl chloride followed by derivatization of the resulting anhydridewith (R)-4-benzyl-2-oxazolidinone (R-OXZ) provides the chiral imide thatcan be hydroxymethylated in a diastereoselective fashion to yield 36.Cyclization to the THF moiety can be accomplished in the presence of aplatinum(II) complex and tris(4-trifluoromethylphenyl)phosphine toprovide 37 as the desired cis imide. It is important to note thatsimilar cyclizations on 36 have afforded an approximately 9:1 mixture ofthe cis:trans THF imide, suggesting the sterics around the THF ringpredispose formation of the cis regioisomer. If significant formation ofthe trans imide does occur, we will separate the isomers and proceedwith both to determine whether the orientation around the THF moietyaffects the anti-Hh activity of this scaffold. Lithium borohydridereduction of 37 to the primary alcohol, followed by tosylation willafford key intermediate 38. The PSZ linker/side chain region will beprepared as described and coupled with 38 to provide the finaldes-triazole PSZ analogue 39.

Example 7: Preparation of Triazolone/Side Chain Region ITZ Analogues

a. ITZ analogues with a modified triazolone ring. ITZ analoguesincorporating both cyclic and acyclic triazolone mimics designed toprobe the sterics, hydrogen bonding, and electronics associated withthis region of the scaffold will be prepared. First, the triazolone willbe substituted with an acyclic urea moiety to determine whether thisregion is amenable to increased flexibility (42). Treatment ofintermediate amine 40 with sec-butyl isocyanate provides thecorresponding urea 41 (Scheme 6). Deprotection of the methyl ether andcoupling to des-triazole intermediate 4 will follow standard protocolsto yield acyclic analogue 42.

Several cyclic analogues will be prepared to address whethermodifications that maintain the more rigid structure of the triazoloneimprove Hh inhibitory activity. First, the N−1 of the triazolone will besubstituted with a carbon to probe the necessity of a hydrogen bondacceptor in this location. Conversion of 40 to the phenyl carbamate andsubsequent cyclization in the presence ofN-(2,2-dimethoxyethyl)butan-2-amine will provide the cyclizedintermediate 43 (Scheme 7, top). Standard protocols will be utilized tounmask the terminal alcohol and couple with 4 to provide analogue 44. Inaddition, the alkene of the triazolone mimic in 44 will be reduced toyield 46, an analogue that will provide information as to whethersaturation of this region has an effect on the Hh inhibitory activity ofITZ (Scheme 6, top). Finally, an analogue that incorporates asix-membered triazolone mimic will be prepared to evaluate how the sizeof the heterocyclic ring affects Hh inhibition. This compound will beprepared by condensing 1,3-dichloropropane with urea 41 (Scheme 7,bottom). Coupling of 47 and 4 will provide the final 6-memberedtriazolone mimic 48 for evaluation.

b. Side Chain Region Analogues. Analogues that incorporatestereochemically defined side chains that mimic the PSZ side chain(49-54) will be prepared. (Scheme 8) These analogues will providefurther SAR information as to how defined stereochemistry of the sidechain affects Hh inhibition. In addition, they will help determinewhether the enhanced Hh inhibitory activity of PSZ in the C3H10T1/2cells is mediated through the hydroxylated side chain. Finally,incorporation of the hydrophilic hydroxyl moiety is predicted to enhancewater solubility compared to Analogue 1. Each of these analogues will beprepared using the standard methodology utilized to synthesize eitherITZ or PSZ as described above and as is known in the art.

Example 8: Preparation of Linker Region ITZ Analogues

A recent report from a research group at Novartis detailed theoptimization of a series of piperizines that ultimately resulted in thegeneration of NVP-LEQ506 as a potent inhibitor of Hh signaling. Based onthe structural similarities between the linker region of ITZ and thecentral core of NVP-LEQ506 (aryl ring-piperizine-aryl ring), and withoutbeing held to theory, it is hypothesized that these two compounds sharea similar binding site. For this reason, the linker region ITZ analogueswill focus on incorporating pyridine, pyrazine, or pyridazine rings intothe scaffold to mimic these moieties in NVP-LEQ506. Not only will theseanalogues provide key SAR for the linker region of the ITZ scaffold, butincorporation of the heterocycles should also have the added benefit ofimproving aqueous solubility.

a) ITZ analogues incorporating heterocycles adjacent to the triazolone.First, analogues that incorporate either a 2-(55) or 3-pyridine (56)ring adjacent to the triazolone will be prepared as previously described(Scheme 9, top). Next, analogues will be prepared that incorporateeither a pyrazine (61) or pyridazine (62) ring as the aromatic moiety inthis location. (Scheme 9, bottom) The synthesis of these analogues willstart with either 2-amino-5-bromopyrazine (67) or6-bromo-3-pyridazinamine (68), which will be converted to the tricycliclinker region intermediates 59-60 via standard conditions. Completion ofthe final analogues will follow those synthetic steps previouslyestablished herein.

b. ITZ analogues incorporating heterocycles adjacent to the triazoleregion. Next, analogues that incorporate either a pyridine (71-72),pyridazine (73), or pyrazine (74) ring as the aromatic moiety adjacentto the triazole region will be prepared (Scheme 10). The synthesis ofthe analogues will start with the requisite commercially availablechloromethoxy heterocycles (63-66), which can be converted to thetricylic linker region intermediates 67-70 via a two-step sequence.Completion of the final analogues will be as described herein.

Example 9: Second Generation Analogues

Based on the SAR and SPR results for the analogues described above,optimal substitutions from each region will be incorporated into asingle scaffold to generate second generation ITZ analogues.Representative examples of compounds that can be explored based on ourSAR/SPR are included below.

Example 10: Triazole Region ITZ Analogues

This series of ITZ analogues will focus on replacing the triazole ringwith a suitable bioisostere to probe whether modifications to thismoiety enhance the inhibitory effects of ITZ on the Hh pathway whileconcomitantly decreasing CYP3A4 inhibition (Scheme 11). These compoundswill include several analogues (75-80) that incorporate aromatictriazole mimics designed to maintain the general size and shapeassociated with the triazole while modifying or removing the N−4associated with the detrimental side effects. In addition, severalanalogues (81-84) that incorporate non-aromatic moieties will beprepared and evaluated to probe whether an aromatic functionality inthis region is essential for Hh inhibition. Synthetic routes to each ofthese intermediates in Scheme 12 have been extensively characterized inthe literature on large scale (>100 g) with minimal chromatographicseparations. To date, multigram quantities of each intermediate havebeen prepared.

Analogues 75 and 81-84 that are appended to the central carbon vianitrogen will be prepared by direct displacement of the bromine inintermediate 85 with the requisite nitrogen nucleophile per the standardsynthesis of ITZ (Scheme 12). By contrast, incorporating the triazolemimics in analogues 76-80 into intermediate 85 is not asstraightforward. Initially, we will explore standard Suzuki and Negishitype cross couplings, to directly append the triazole mimics to 85. Inaddition, we will also explore coupling the corresponding aryl Grignardreagents to 85 in the presence of catalytic quantities of a copper salt.In the event that direct coupling to the bromo ketal is unsuccessful, adifferent synthetic route will be utilized to prepare these analogues(Scheme 13). Commercially available 2,4-dichlorobenzaldehyde, 89, willbe converted to the corresponding epoxide, 90, through recentlypublished protocols. Epoxide opening at the less substituted carbon withan aryl magnesium bromide followed by reduction of the secondary alcoholwill provide the ketone intermediate 91 that can be directly convertedto ketal 92 via standard ITZ synthesis conditions. Coupling of thesetriazole region fragments to intermediate 88 will provide final ITZanalogues 75-84 for evaluation.

The second series of triazole region ITZ analogues that will be preparedfocuses on truncating the triazole region to determine the essentialpharmacophore for this portion of the compound. We will take aconservative and systematic approach to modifying this region to providea better understanding of how each functionality contributes to Hhinhibition (Scheme 14). Analogues that will be prepared include anon-substituted phenyl analogue (93), a des-phenyl analogue (94), ades-triazole analogue (95), and an analogue that removes both the phenyland triazole rings (96). In addition, intermediate 88 will be evaluatedto determine the general necessity of the triazole region.

Example 11: In Vitro Hh Inhibitory Activity of ITZ Analogues

The ITZ analogues will be screened in a panel of biochemical andcell-based assays to evaluate their potential as anti-MB agents thattarget Hh signaling (FIG. 10). Initially, the analogues will be screenedin the following three assays (1) down-regulation of endogenous Gli mRNAin C3H10T1/2 cells at 1 and 0.5 μM, (2) a BBB-PAMPA (Parallel ArtificialMembrane Permeability Assay) to predict CNS penetration, and (3)P-gp/BCRP substrate assays. These assays will not only provide an earlystage description of SAR for Hh inhibition, but they will also providevaluable information as to how the structural modifications affectessential PK properties for the ITZ scaffold. Compounds that reduce Gliexpression below 10% at 1.0 μM and 35% at 0.5 μM will be assessed fortheir ability to inhibit Hh signaling in a concentration-dependentfashion in multiple, distinct cellular models, including C3H10T1/2 andHh-dependent MB cells. (Example 13). ITZ analogues that advance to thestudies described in Example 13 will also be evaluated in a parallelseries of assays to generate a preliminary pharmacokinetic profile forthese compounds (Example 14). As these assays are well-established inthe literature, only brief descriptions will be provided herein. Whereappropriate, ITZ, PSZ, and/or GDC-0449 will be used as positivecontrols. GraphPad Prism software is used for all graphing andstatistical analysis. Specifically, IC₅₀ and GI₅₀ values are calculatedvia non-linear regression analysis and values represent mean±SEM for atleast two separate experiments performed in triplicate. Two-sidedStudent's t-tests are utilized to determine significance between treatedand non-treated samples when a single concentration is evaluated.One-way ANOVA followed by Tukey's test is utilized to determinesignificance related to the following: (1) response to multipleconcentrations of the same analogue and (2) response to multipleanalogues (including controls) at a single concentration.

Background: The last twenty years have seen a concerted effort todevelop computational models and statistical equations that can beutilized during the early stages of drug design to accurately predictwhether a small molecule will penetrate the BBB. Common parametersassociated with these models include molecular weight (MW),lipophilicity (log P or log D), total polar surface area (tPSA),H-bonding properties, and 3D molecular interaction fields. While many ofthese equations have proven useful at predicting BBB permeability, noneof them is absolute and all demonstrate several shortfalls. On average,the correlations between predicted BBB penetration and experimentallydetermined brain concentrations are good, but not absolute (r²values=0.72-0.92) and correct classifications are generallyapproximately 85%. These models predict the probability that a smallmolecule will passively diffuse through the BBB, but they cannot accountfor compounds that are actively transported into the CNS or those thatare actively effluxed from the brain. In addition, statistical equationscan only predict total drug concentration and not the free drugconcentration after plasma protein binding. Finally, many disease statesare known to affect BBB integrity and can actually result in enhancedpermeability that would not be predicted from any of the standardcomputational models.

As noted above, ITZ increased survival in an orthotopic model ofHh-dependent MB following oral administration, providing strong evidencethat it accumulates in the brain in efficacious concentrations withinthe context of murine models of intracranial MB. Another parameter thatmust be considered for developing drugs that target intracranial tumorsis to what extent they are actively effluxed from the brain. Currently,there are two multidrug transporters that are recognized as the majorefflux pumps in the BBB; P-glycoprotein (P-gp, ABCB1 or MDR1) and breastcancer resistance protein (BCRP, ABCG2). Two separate studies evaluatingbrain accumulation of ITZ demonstrated that concentrations of the drugin the brain were significantly increased in mdr1a knockout mice or whena P-gp inhibitor (verapamil) was co-administered, suggesting ITZ is asubstrate for P-gp efflux. While ITZ has been identified as a modestinhibitor of BCRP (IC₅₀ approximately 1 μM), studies to determinewhether it is a substrate for BCRP-mediated efflux have not beenreported.

With all of this as context, it is clear that BBB penetration and brainaccumulation are highly dependent on multiple factors, many of which arespecific to the individual scaffold of the drug. Determining the factorsthat contribute both to BBB penetration and efflux of the ITZ scaffoldis of equal importance to Hh inhibitory ability with respect todesigning and developing analogues that exhibit improved activityagainst intracranial tumors. For this reason, we will determine both SARfor Hh inhibition and SPR for brain penetration and accumulation duringthe early stages of in vitro analysis for the ITZ analogues.

Initial in vitro assays for Hh inhibition and BBB penetration andaccumulation. With respect to Hh inhibition, all analogues will beevaluated for their ability to down-regulate endogenous Gli mRNA in theHh-dependent C3H10T1/2 cell line at two concentrations (1 μM and 0.5μM). Hh inhibition for both ITZ and 1 are comparable at these twoconcentrations (approximately 98% and 70%, respectively) and they willprovide an appropriate measure of the inhibitory activity of theadditional analogues. This assay will be performed as known in the artand in the same manner utilized for the preliminary data reported for 1.

Each analogue will also be evaluated in assays to develop SPR for theability of the scaffold to penetrate the BBB and accumulate in thebrain. First, a standard BBB-PAMPA assay will be used to predict theability to passively diffuse across the BBB. In this method, anartificial membrane that has been designed to mimic the BBB isimmobilized on a filter between a donor and acceptor compartment. ITZ oranalogue (10 μM) is added to the donor compartment and following a 30min incubation period, compound concentrations in both compartments arequantified via LC/MS/MS and this data will be utilized to determine theapparent permeability (Papp) as a predictor of BBB penetration. StandardP-gp/BCRP transporter assays are also performed to determine to whatextent the ITZ analogues are actively effluxed from the brain. For theseassays, Madin Darby canine kidney cells (MDCK) are transfected witheither P-gp or BCRP and grown to confluence on filter inserts in atranswell apparatus analogous to that used for the PAMPA assay. Analogue(10 μM) is added to either the donor or acceptor compartment andfollowing a 60 min incubation period, compound concentrations in bothcompartments are quantified via LC/MS/MS. These data will be utilized tocalculate an efflux ratio. Should the results suggest an analogue is asubstrate for either transporter, the assay will be repeated in thepresence of a P-gp (verapamil) or BCRP (Elacridar) inhibitor todetermine whether efflux is suppressed. The fluorescent dye luciferyellow will be utilized to ensure membrane integrity in the transporterassays. The BBB-PAMPA assay and both transporter assays will beperformed in collaboration with the Sanford-Burnham ExploratoryPharmacology Core Facility.

In preliminary studies with PSZ in the BBB-PAMPA assay, an apparentpermeability (Pap) of 46 was measured, suggesting that PSZ is moderatelypermeable. For reference, verapamil is considered highly permeable witha Papp of 170; corticosterone is moderately permeable with a Papp of 15,and theophylline is impermeable with a Papp of 0.15.

In addition, it was tested whether posaconazole is a substrate of P-gpor BRCP, and posaconazole was not a substrate of either, suggesting thatif it gets in the brain, it is not actively removed by known mechanisms.

Example 12: Secondary In Vitro Assays for Concentration-DependentInhibition of Hh Signaling

Analogues that reduce Gli mRNA expression comparably to ITZ and 1 at 1(<10%) and 0.5 (<35%) μM and effectively penetrate the BBB-PAMPA filterwill be further evaluated for their ability to down-regulate endogenousGli and Ptch in C3H10T1/2 cells in a dose-dependent fashion to establishan IC₅₀ value for Hh inhibition in this cell line. These analogues willalso be evaluated for their effects on Hh pathway signaling in thepresence of a mutated form of SMO (D477G) that confers resistance tomultiple Hh pathway inhibitors including GDC-0449. For this assay, thewild-type and D477G Smo constructs will be transiently transfected intoC3H10T1/2 cells and inhibition of pathway signaling assessed aspreviously described. Compounds that exhibit IC₅₀ values less than 200nM and retain potent Hh inhibition in the presence of mutant Smo will beconsidered for further evaluation in the secondary analyses describedbelow. Of note, the benchmarks that have been set for advancingcompounds to the next phase are primarily based on the activity of ITZand 1; however, these benchmark metrics will be continually evaluated toensure that an appropriate number of analogues are being advanced to thenext stage.

ITZ analogues that exhibit Hh inhibition in C3H10T1/2 cells per theguidelines established above will be further evaluated for their abilityto modulate Hh signaling in a dose-dependent fashion in cells fromHh-dependent models of MB, using protocols previously established in theart. MB cells will be isolated from conditional patched knockout(Math1-CreER^(T2);Ptch^(flox/flox)) mice and cultured as described inthe art. ITZ analogues will be evaluated for their ability todown-regulate endogenous Gli expression and for their effects on cellproliferation (³H-thymidine incorporation and cell cycle analysis),differentiation (expression of Math1, TuJ1 and MEF2D) and survival(cleaved caspase expression, Annexin V/propidium iodide staining).

To further explore the efficacy of ITZ analogues against human MB,compounds will be evaluated in patient-derived xenograft (PDX) cells,which represent unique tools to study Hh pathway inhibitors for theiranti-MB activity. PDX lines are created by isolating cells fromsurgically-resected human MB tissue and directly implanting them intothe cerebellum of immunocompromised (NOD-SCID-IL2Rgamma or NSG) mice.Cells are passaged from mouse to mouse, and are never grown in cultureexcept for short-term experiments. Tumor cells propagated in this mannerretain many of the molecular and cellular properties of the tumors fromwhich they were derived. A panel of MB PDX lines has been established inthe art, including several that exhibit Hh pathway activation. For theseexperiments, tumor bearing mice will be sacrificed and tumor cellsdissociated and plated in vitro. The effect of ITZ analogues on Gliexpression, proliferation, and survival of the PDX cells will beevaluated using the assays described above. It is important to note thatwhile the PDX cells were directly isolated from human medulloblastomatumors, we have no information that would allow us to identify theindividuals from whom the samples were obtained.

Example 13: Preliminary Pharmacokinetic Studies

Understanding the pharmacokinetic (PK) profile of a potential drugcandidate during the early stages of its development has become a keystrategy for improving its drug-like properties. The final in vitrostudies that will be performed will provide an evaluation of thefollowing key PK parameters: solubility, stability (metabolic andchemical), and intestinal permeability. The results from these studieswill provide an overall PK profile for each class of analogues, aid indeveloping formulations for IP and oral dosing, and predict oralbioavailability.

a. Solubility. ITZ was initially available in several capsuleformulations; however, the plasma and tissue concentrations followingadministration of these capsules were highly variable and dependent onnumerous patient-specific parameters. Studies on inclusion complexesbetween ITZ and 2-hydroxypropyl-β-cyclodextrin (2β-CD) demonstrated thatthese complexes significantly enhanced both solubility and oralbioavailability of ITZ. These initial results led to the development ofan oral solution of ITZ (Sporanox®) containing ITZ (10 mg/mL) and 2β-CD(400 mg/mL) with a pH of 2, which demonstrates an improved oralbioavailability and more consistent tissue distribution and plasmaconcentrations. Many of the modifications of the ITZ scaffold will alsoaffect the log P and aqueous solubility of the ITZ scaffold (i.e.,removal of the triazole, incorporation of hydroxyl(s) in the side chainand pyridine rings in the linker region). First, standard protocols willbe utilized to determine kinetic solubility, a common early stagemeasure of aqueous solubility. The standard benchmark for kineticsolubility is >60 μg/mL as compounds that obtain this value generallydemonstrate enhanced bioavailability following both oral and systemicdosing. ITZ analogues with promising potency that do not reach thissolubility threshold in water will be prepared as inclusion complexeswith 2β-CD per previously published protocols to enhance solubility inpreparation for the in vivo studies.

b. Chemical and metabolic stability. Next, a series of in vitro assayswill be performed to assess the chemical and metabolic stability of theoptimal ITZ analogues. Standard procedures will be utilized to evaluatechemical stability in simulated gastric fluid (pH 1.2), simulatedintestinal fluid (pH 7.4), and water. In addition, if complexation isnecessary to achieve the benchmark solubility, chemical stability willalso be assessed in this solution. Standard assays to characterize theoverall metabolic stability and identify major metabolites for our ITZanalogues will also be performed. Numerous HPLC and LC/MS assays foranalyzing clinical samples containing ITZ and its metabolites have beendeveloped. For this reason, these previously optimized conditions (HPLCmethod, concentration, incubation time, detection method, etc.) will beutilized as a basis for these assays. ITZ analogues will be incubated inpooled human or rat liver microsomes (BD Biosciences) and samplesanalyzed at various time points for metabolic stability. Resultingmetabolites will be characterized via LC/MS and NMR and majormetabolites will be synthesized and characterized via the methodsdescribed for Analogue 1 to determine their biological activity withrespect to the Hh signaling pathway. Each ITZ analogue will also beevaluated for its ability to inhibit a panel of major hepaticdrug-metabolizing CYP450 enzymes (1A2, 2A6, 2C9, 2C19, 2D6, and 3A4).Several of these enzymes are known to be inhibited by various azolesincluding ITZ (2A6, 2C9, and 3A4). For this assay, ITZ or analogue willbe incubated with human CYP450 Supersomes™ (BD Biosciences) as describedin the art and evaluation of inhibitory activity will be assessed withthe P450-Glo™ Assay Kit (Promega) via the manufacturer's protocols.

c. Intestinal permeability. Finally, Caco-2 cell monolayers will beutilized as a predictor of intestinal absorption and oralbioavailability. Technological advances in assays for oral permeabilityhave led to the development of commercially available assay kits thatallow for drug transport studies in a 96-well plate format (MultiScreen®Caco-2 Assay System). For these assays, analogues will be evaluated at10 μM following the manufacturer's suggested protocol. Followingincubation, analogue quantification in both the apical and basolateralchambers will be analyzed via LC/MS/MS and this data will be utilized todetermine the apparent permeability (Papp) as a predictor of intestinalabsorption. The small molecule mannitol will be used as a control formonolayer integrity.

It is anticipated that the in vitro studies will result in theidentification of SAR for several regions of the ITZ scaffold withrespect to selective inhibition of Hh signaling in C3H10T1/2 andHh-dependent MB cells. In addition, the solubility, stability, andpermeability studies will provide essential early stage PK parametersfor developing the ITZ scaffold as an anti-MB chemotherapeutic. Takentogether, these results will identify ITZ analogues with potent anti-Hhactivity and improved drug-like properties for in vivo studies. Based onour identification of Analogue 1 as a potent analogue of ITZ and theinherent activity of ITZ in many of these assays, it is anticipated thatmultiple analogues will meet the potency benchmarks. In addition, thefact that ITZ increased survival in an orthotopic model of MB stronglysuggests that these analogues will be able to cross the BBB at effectivedoses, irrespective of the in vitro results.

Example 14: Evaluation of ITZ Analogues in Murine Models of Hh-DependentMB

The compounds selected on the basis of the in vitro assays of Examples12-14 will first be evaluated for their ability to inhibit the growth ofHh-dependent MBs in flank allografts (Example 15). This allograft modelwill not only allow for determination of the anti-Hh and anti-MBactivity of ITZ and its analogues irrespective of brain penetration, butwill also provide preliminary information for the treatment of otherHh-dependent peripheral cancers. Analogues that exhibit activity inflank allografts will be evaluated for their ability to cross the BBBand accumulate in the brain (Example 16). Compounds that reachefficacious concentrations in the brain will then be evaluated for theirefficacy in intracranial tumors (Example 17). Finally, ITZ analoguesthat exhibit potent in vivo activity will be evaluated in a series ofstudies to determine their propensity to induce resistance as aprospective step to improve the overall drug discovery process of ITZanalogues as anti-MB agents that target the Hh signaling pathway(Example 18).

Background and Preliminary Studies. Mouse models are invaluable toolsfor studying tumor biology and developing novel therapeutics. Mice withmutations in Ptch have been utilized to develop tumors that closelymimic human Hh-associated MB. These animals have been used to study manyaspects of Hh tumor biology, including the cell of origin, the earlystages of tumorigenesis, and the genetic lesions that cooperate withloss of Ptch to induce tumor formation. In addition, Ptch mutant micehave been used as a platform for preclinical testing of Hh pathwayinhibitors and other therapeutic agents. Most importantly for thepurposes of the proposed studies, the tumors that develop in Ptch^(−/−)mice harbor mutations upstream of Smo. As ITZ has been proposed toinhibit Hh signaling via direct binding to Smo, these mice represent anexcellent model to study the anti-Hh and anti-cancer properties of ITZanalogues in vivo.

Mouse models of MB, including genetically engineered mouse (GEM) andpatient-derived xenograft (PDX) models, have been used for preclinicalstudies of therapeutic agents and treating MB in vivo. As a proof ofprinciple experiment for evaluating Hh pathway inhibitors in thesemodels, a recent study was conducted using LDE-225, a small molecule Hhpathway inhibitor currently in multiple clinical trials for thetreatment of several types of human cancer. For these studies, tumorcells from a PDX line (with a mutation in PTCH1) were infected withluciferase viruses and implanted into the cerebellum ofimmunocompromised NSG mice. After four weeks, daily oral gavage wasinitiated with LDE-225 or vehicle and animals were monitored bybioluminescent imaging. LDE-225 caused tumor regression and prolongedsurvival (FIGS. 11A and 11B). The results from these studies verify thatthis model is suitable for analysis of Hh pathway inhibitors anddemonstrate that the investigators have the expertise necessary toconduct the proposed experiments.

Anti-MB studies in flank allograft models of Hh-dependent MB. Evaluatingthe ITZ analogues in flank allografts is an important first step toprobing the in vivo activity of the compounds. These studies willprovide information as to the ability of the analogues to inhibitpathway signaling and decrease tumor growth in an Hh-dependent murinemodel of MB, irrespective of their ability to cross the BBB andaccumulate in the brain. In addition, comparing the effects of the ITZanalogues after IP and oral dosing will provide preliminary informationwith respect to the oral bioavailability of the analogues. Finally,tumors that do not respond to ITZ or analogue treatment will be avaluable source of information for determining the potential resistancemechanisms that are induced for this particular scaffold (see Example17). It is important to note that the initial doses chosen for the invivo studies are based on the doses for which ITZ has demonstratedpotent anti-Hh and anti-MB activity in vivo. These doses will bemodified as needed should the analogues prove more or less active thanITZ in vivo.

For the flank allografts, Math1-CreER^(T2);Ptch^(flox/flox) tumor cellswill be infected with viruses encoding a GFP-luciferase fusion protein(pGreenFire, System Biosciences) that allows FACS sorting as well asbioluminescent imaging of tumor cells. Infected (FACS-sorted GFP+) cellswill be mixed 1:1 with growth factor-reduced matrigel and implanted intothe flanks of Nu/Nu mice (2×10⁶ cells/host). Tumor growth will bemonitored using calipers as well as bioluminescence imaging. When tumorsreach a volume of approximately 100 mm³, animals with comparably sizedtumors will be segregated into treatment groups (ITZ, ITZ analogue, orvehicle) and treatment will be initiated. Based on survival poweranalysis, assuming the analog inhibitors work at least as well as otherHh antagonists, 20 mice per group will be needed for 80% power(p-value=0.05). Animals will be given daily intraperitoneal (i.p. 50mg/kg) or oral (100 mg/kg) doses and sacrificed when the largest tumorin the treatment group reaches 2 cm in any dimension. After sacrifice,tumors will be collected, weighed, and photographed to determine theeffects of compounds on overall tumor size. For a subset of animals fromeach group, tumor tissue will be processed for RNA isolation, and Gli1levels will be assessed by qPCR. For an additional set of animals,tissue will be fixed, sectioned, and stained with markers ofproliferation (Ki67), differentiation (Math1, TuJ1, MEF2D) and apoptosis(cleaved caspase).

Example 15: In Vivo Blood Brain Barrier Penetration

Analogues that exhibit anti-Hh and anti-MB effects in flank allograftswill be evaluated to determine their ability to cross the BBB andaccumulate in the brain. Initially ITZ, analogue, or fluconazole (anazole antifungal known to cross the BBB) will be administered via eitheri.p. injection or oral gavage to tumor-bearing mice to identify how thestructural modifications affect BBB penetration and accumulation. Thedose to be used for these studies will be determined based on theresults of the allograft studies in Example 15. Animals (4 mice per timepoint) will be sacrificed 1, 3, 6, and 12 hrs post-injection, and plasmaand brain tissue will be frozen and submitted to the EPC for analysis.Drug concentrations in plasma and brain will be measured by LC/MS/MS andratios between the two determined. If analogues achieve brainconcentrations corresponding to those determined to be effective in thein vitro studies, these compounds will immediately move to intracranialtumor studies described below. In the event that BBB accumulation islow, the study will be repeated and the analogues will be coadministeredwith either verapamil (5 mg/kg), an inhibitor of P-gp previously shownto significantly enhance brain concentrations of ITZ, or Elacridar (10mg/kg), a dual inhibitor of both P-gp and BCRP. Comparing the resultsfrom both transport inhibitors will determine whether ITZ efflux ismediated by one or both of the major multi-drug transporters in vivo.

Example 16: Anti-MB Studies in Intracranial Tumors

Agents that are determined to be effective in flank allografts andaccumulate at therapeutic concentrations in the brain will be evaluatedfor their effect on the growth of intracranial tumors. For thesestudies, GFP-luciferase infected Math1-CreER^(T2);Ptch^(flox/flox) tumorcells will be sorted and implanted into cerebella of NSG mice. Whentumors are detectable by bioluminescence (approximately 4 weeks aftertransplantation), animals with comparably sized tumors will besegregated into treatments groups (ITZ, ITZ analogues, or vehicle) andtreatment will be initiated. Animals (20 mice per treatment group) willbe given daily i.p. (50 mg/kg) or oral (100 mg/kg) doses of eachcompound. One cohort of mice will be sacrificed after 30 days and tumortissue analyzed as described above to evaluate the effects of ITZ andanalogues of Gli1 expression, proliferation, differentiation, andapoptosis. A second cohort of mice will be followed until onset ofsymptoms to assess the effects of analogues on animal survival.

Example 17: Prospective Resistance Studies

As noted above, resistance has developed for each of the small moleculeHh pathway inhibitors that have been extensively studied in preclinicalmurine models of MB; therefore, it is useful to explore the potential ofboth ITZ and its analogues to promote tumor resistance. These studieswill be performed in combination with the in vivo tumor analysesdescribed in Examples 15 and 16 above. Specifically, tumors from bothstudies that do not respond to therapeutic treatment will be assessedfor the resistance mechanisms previously identified for GDC-0449,NVP-LDE225, and IPI-926 following the protocols used for thesecompounds. Genomic DNA will be extracted from the tumors, amplified viaPCR, and all Smo exons will be sequenced to identify missense pointmutations. In addition, mRNA and protein samples will be isolated fromthe tumors and analyzed for mutation or amplification of key Hh pathwaycomponents downstream of Smo (Sufu, Gli1, Gli2, Gli3, etc.) that couldpotentially contribute to the development of resistance. Finally, tumorsamples will be evaluated for the up-regulation of P-gp and BCRP (mRNAand protein) to determine whether developed resistance could be linkedto increased analogue efflux.

The studies described in Examples 15-18 will provide essential dataregarding the potential of ITZ analogues to inhibit Hh-dependent MB invivo. First, they will identify to what extent ITZ and its analoguescross the BBB in animal models of MB and determine whether addition of atransport inhibitor may increase uptake. Second, by directly comparingthe analogues in flank allografts and intracranial tumors we will gainadditional insight into whether the BBB affects the anti-Hh and anti-MBproperties of the compounds. Finally, we will learn whether resistanceto the ITZ scaffold develops in a fashion similar to that seen for theother Hh pathway inhibitors that have entered clinical trials for MB.

Example 18Angiogenesis Inhibition Assays

HUVEC Cell Viability and Proliferation. HUVEC cell viability andproliferation will be assessed by measuring cellular metabolic activityusing MTT (viability) or MTS (proliferation) according to themanufacturer's instructions. HUVECs (3000 cells/well) are seeded in a96-well plate and allowed to attach overnight. Cells are treated withvarying concentrations of ITZ or Analogue; viability is assessed after24 h with MTT and proliferation was assessed after 72 h with MTS. Forviability, the final value is a corrected optical density (570 nm-690nm). All viability and GI50 values are from a minimum of three separateexperiments performed in triplicate.

Tube Formation Assay. Matrigel (BD Biosciences) is diluted with DMEM to3 mg/ml and used to coat the wells of a 24-well tissue culture dish.Plates are incubated at room temperature for 30 min until the matrigelsolidifies. HUVECs are suspended in M199 media with 1% FBS andpenicillin/streptomycin and 40,000 cells are added to each well. Platesare incubated at 37° C. for 1 h to allow HUVECs to attach to thematrigel. Cells are treated with 100 ng/ml VEGF (R&D systems) andcontrol (DMSO) or varying doses of ITZ and Analogue and incubated for 12h. Phase contrast images of each well (3/well) are taken on an invertedmicroscope and quantified using Image J software (NIH).

Example 19: Preliminary Testing of Additional Analogues

Additional analogues have been synthesized and characterized in theassays described above.

IC₅₀ (μM) IC₅₀ (μM) IC₅₀ (μM) IC₅₀ (μM) IC₅₀ (μM) Name StructureGli1-C3Hs Ptch1-C3Hs Gli1-DAOYs Gli1-ASZs Gli1-M2s ITZ

0.07 ± 0.02 0.20, 0.33  0.6 ± 0.08 0.14 ± 0.02 0.30, 0.52 PSZ

 0.01 ± 0.005 ND 0.89 ± 0.1  0.54 ± 0.05 ND Amd5-54 (Analogue 1)

0.14 ± 0.04 ND ND 0.17 ± 0.04 ND Itz-psz #1 (S-)

0.45 ± 0.09 ND ND 0.15 ± 0.01 ND Itz-psz #2 (R-)

0.18 ± 0.02 ND ND 0.10 ± 0.01 ND

The data from these compounds demonstrates that modifications to thetriazole region of ITZ and PSZ, as well as the side chain region do notaffect the ability of the scaffold to inhibit Hh signaling. Each ofthese compounds demonstrates potent in vitro activity in Hh-dependentmouse embryonic fibroblasts and cultured MB (DAOY) and BCC (ASZ) celllines.

Example 20: Identification of Key ITZ Features Required for Hh Signalingand Angiogenesis Materials and Methods

General Information.

Starting materials were purchased from Sigma-Aldrich or FisherScientific. ACS grade methanol, ethyl acetate, toluene, anhydrous DMF,NMP, and DMSO were purchased from Fisher Scientific or Sigma-Aldrich.ITZ analogue 9a was purchased from Toronto Research Chemicals. Allreactions were run under an argon atmosphere. NMR data was collected ona Bruker AVANCE 500 MHz spectrometer and analysis performed usingMestReNova. HRMS data was analyzed at the Mass Spectrometry Facility atthe University of Connecticut by Dr. You-Jun Fu. FT-IR analysis wasperformed on a Bruker Alpha Platinum ATR instrument using OPUS software(v 7.2). The preparation of previously characterized ITZ intermediatesfollowed known procedures with minor modifications. X-ray crystals wereprepared using vapor diffusion techniques (pentanes:chloroform) andanalysis performed by Dr. Victor Day at the Small-Molecule X-rayCrystallography Lab at the University of Kansas on a Bruker MicroStarmicrofocus Cu rotating anode generator with two CCD detectors or aBruker Apex II CCD detector equipped with Helios multilayer opticsinstruments. Mercury (v3.0) software was used to visualize X-raystructural analysis. All ITZ analogues evaluated in the biologicalassays (1a-25a) were greater than 95% pure based on the HPLC methodsdescribed below.

Purity Analysis of Final Analogues.

It is important to note that final ITZ analogues 1a-11a were synthesizedand evaluated as stereoisomeric mixtures. For this initial series, wedid not separate individual stereoisomers nor did we determine theratios of cis:trans dioxolanes produced in the ketalization reaction.These mixtures are reflected in the ¹H and ¹³C NMR characterization datadescribed below. Purity analysis for all final analogues was determinedvia one of the methods described below.

Method A.

ITZ analogues were dissolved in HPLC grade MeCN and injected (20 μl of a1 mM soln) into an Agilent Manual FL-Injection Valve (600 bar) on anAgilent 1100/1200 Series HPLC equipped with an Agilent Eclipse Plus C18(4.6×100 mm) column and Agilent 1100 Series Photodiode Array Detector.The mobile phase consisted of 60% MeCN:40% H₂O for analogues containingthe triazole moiety and 70% MeCN:30% H₂O for des-triazole analogues. Allanalogues were run at a flow rate of 1.0 mL/min for 20 mins and puritywas assessed at 254 nm.

Method B.

ITZ analogues were dissolved in HPLC grade MeCN and injected (20 μl of a1 mM soln) into an Agilent HPLC system coupled to an Agilent ESI singlequadrupole mass spectrometer equipped with a Kinetix C18 (150×4.6 mm)column and an Agilent G1315 diode array detector. The mobile phaseconsisted of 70% MeCN:30% H₂O. All analogues were run at a flow rate of0.7 mL/min for 30 mins and purity was assessed at 254 nm.

Previously Characterized ITZ Intermediates.

Common ITZ linker region (28a-32a and 68a), dioxolane region (53a-56a,59a, 62a-63a), side chain (35a-36a), and coupled intermediates wereprepared primarily as described previously for the ITZ scaffold with theminor modifications. Procedures and characterization for newly reportedkey intermediates and all final analogues is provided below.

First Generation ITZ Intermediates and Final Analogues (1a-11a).

1-(1H-1,2,4-triazol-1-yl)propan-2-one (51a). A solution of chloroacetone(47a) (1.72 mL, 21.6 mmol), 1H-1,2,4-triazole (50a) (2.98 g, 43.2 mmol),NaHCO₃ (2.89 g, 34.5 mmol), and toluene (100 mL) were heated to reflux(110-120° C.) for 3 h. The reaction vessel was cooled to −20° C. for 12h. The resulting precipitate was filtered, dissolved in H₂O, andextracted with EtOAc (50 mL×3). The organic layer was collected, washedwith saturated sodium chloride (100 mL), and dried (Na₂SO₄). The solventwas evaporated and the crude product was purified via columnchromatography (SiO₂, 0-3% MeOH in DCM) resulting in a yellow oil (600mg, 22.2%). ¹H NMR (500 MHz, CDCl₃) δ 8.01 (s, 1H), 7.76 (s, 1H), 4.90(s, 2H), 2.00 (s, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 199.58, 151.41,144.15, 57.52, 26.63. DART-HRMS: m/z calcd. for C₅H₇N₃O [MH]⁺, 126.0667;Found: 126.0691. IR (solid) vmax: 3122, 2935, 2241, 1728, 1507, 1352,1273, 1137, 1115, 727, 677, 646.

1-phenyl-2-(1H-1,2,4-triazol-1-yl)ethanone (52a). A solution of2-bromoacetophenone (48) (2.0 g, 10.0 mmol), 1H-1,2,4-triazole (50a)(1.4 g, 20.1 mmol), NaHCO₃ (1.3 g, 16.1 mmol), and toluene (100 mL) wereheated to reflux (110-120° C.) for 3 h. The reaction vessel was cooledto −20° C. for 12 h. The resulting precipitate was filtered, dissolvedin H₂O, and extracted with EtOAc (3×50 mL). The organic layer wascollected, washed with saturated sodium chloride, and dried over sodiumsulfate. The solvent was evaporated and the crude product was purifiedvia column chromatography (SiO₂, 0-3% MeOH in DCM) resulting in a yellowsolid (1.2 g, 64.1%). ¹H NMR (500 MHz, CDCl₃) δ 8.28 (s, 1H), 8.04 (s,3H), 7.70 (s, 1H), 7.57 (s, 2H), 5.71 (s, 2H). ¹³C NMR (126 MHz, CDCl₃)δ 190.84, 152.02, 145.07, 134.69, 129.30, 128.25, 55.19. DART-HRMS: m/zcalcd. for C₁₀H₉N₃O [MH]⁺, 188.0824; Found: 188.0822. IR (solid) vmax:3113, 3062, 2992, 2953, 1697, 1596, 1504, 1450, 1345, 1225, 1207, 1135,1021, 888, 752, 687, 675, 656, 634.

(2-((1H-1, 2,4-triazol-1-yl) methyl)-2-methyl-1, 3-dioxolan-4-yl) methyl4-methylbenzenesulfonate (57a). Ketone 51a (1.4 g, 11.9 mmol) and 56a(2.8 g, 11.5 mmol) were added to a dry round bottom flask. Anhydroustoluene (10 mL) was added and the mixture was cooled to 0° C. at whichtime TfOH (4.0 mL, 45.9 mmol) was added dropwise with a glass syringe.The solution was stirred at RT for 60 h. The mixture was diluted with 50mL of EtOAc and slowly added to a solution of K₂CO₃ (5 g) in water (40mL). The aqueous layer was washed with EtOAc (3×50 mL) and the organiclayers were combined, dried (Na₂SO₄), filtered, and concentrated. Thecrude product was purified via column chromatography (SiO₂, 0-10% MeOHin DCM) (200 mg, <10%). ¹H NMR (500 MHz, CDCl₃) δ 8.10 (d, J=8.3 Hz,1H), 7.86 (s, 1H), 7.81-7.75 (m, 2H), 7.37 (m, 2H), 4.39-4.24 (m, 3H),4.06 (m, 1H), 4.03-3.92 (m, 1H), 3.86 (m, 1H), 3.83-3.74 (m, 1H), 3.69(m, 1H), 3.53 (m, 1H), 2.46 (d, J=5.7 Hz, 3H), 1.32 (d, J=5.9 Hz, 3H).¹³C NMR (126 MHz, CDCL³) δ 152.04, 151.89, 145.75, 144.96, 132.89,130.45, 130.39, 128.40, 74.56, 74.07, 68.83, 68.68, 67.17, 67.03, 55.93,55.88, 23.77, 22.66, 22.09. DART-HRMS: m/z calcd. for C₁₅H₁₉N₃O₅S [MH]⁺,354.1124; Found: 354.1124. IR (solid) vmax: 3007, 2989, 2916, 2894,1594, 1557, 1359, 1172, 1093, 1035, 961, 873, 724, 662, 563.

(2-((1H-1,2,4-triazol-1-yl)methyl)-2-phenyl-1,3-dioxolan-4-yl)methyl4-methylbenzenesulfonate (58a). Crude dioxolane 58a was prepared througha procedure analogous to that described above for 57a. Followingextraction with EtOAc, the combined organic layers were dried (Na₂SO₄),filtered, and concentrated to ˜70 mL EtOAc. A solution of TsOHmonohydrate (2.0 g) in EtOAc (13 mL) was slowly added at RT. The productprecipitated as a salt and was filtered (3.9 g). The salt was dissolvedin aqueous saturated K₂CO₃ (100 mL) and washed with DCM (3×100 mL). Theorganic layers were combined, dried (Na₂SO₄) and used without furtherpurification (62%). ¹H NMR (500 MHz, CDCl₃) δ 8.02 (s, 1H), 7.77-7.70(m, 3H), 7.40-7.27 (m, 8H), 4.42 (d, J=1.4 Hz, 2H), 4.22-4.13 (m, 1H),3.75 (m, 2H), 3.61 (m, 1H), 3.47 (m, 1H), 2.42 (s, 3H). ¹³C NMR (126MHz, CDCl₃) δ 151.12, 145.23, 138.05, 132.33, 129.98, 129.27, 128.62,127.94, 125.64, 108.44, 73.42, 68.52, 66.55, 55.71, 21.65. DART-HRMS:m/z calcd. for C₂₀H₂₁N₃O₅S [MH]⁺, 416.1280; Found: 416.1263. IR (solid)vmax: 3311, 3111, 2992, 2953, 2893, 1596, 1509, 1448, 1349, 1336, 1232,1172, 1043, 968, 811, 736, 699, 611, 551.

(2-(2,4-dichlorophenyl)-2-methyl-1,3-dioxolan-4-yl)methyl4-methylbenzenesulfonate (61a). To a solution of2′,4′-dichloroacetophenone (60a) (20 g, 0.1 mol) in toluene (80 mL) wasadded glycerol (11 g, 0.12 mol) followed by a catalytic amount ofp-toluenesulfonic acid monohydrate (475 mg, 2.5 mmol). A Dean-Stark trap(10 mL trap filled with 8 mL toluene) and condenser were fitted atop thereaction flask. The solution was refluxed for 48 h. Upon cooling, themixture was diluted with EtOAc and washed sequentially with saturatedsodium bicarbonate (3×100 mL), water (2×100 mL), and saturated sodiumchloride (100 mL). The organic layer was dried (MgSO₄), filtered,concentrated, and purified by column chromatography (SiO₂, 3:1 Hex:EtOAc) to yield 61a as a clear oil in excellent yield (88%). ¹H NMR (500MHz, CDCl₃) δ 7.78 (d, J=8.2 Hz, 1H), 7.47 (m, 1H), 7.36-7.31 (m, 2H),7.31-7.27 (m, 1H), 7.18-7.14 (m, 1H), 4.16 (m, 1H), 4.10-4.00 (m, 2H),3.89-3.77 (m, 1H), 3.68 (m, 1H), 2.43 (s, 1H), 2.42 (s, 2H), 1.66 (d,J=1.0 Hz, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 144.98, 13 7.50, 134.44,132.50, 130.95, 130.69, 129.80, 129.74, 128.58, 128.11, 127.81, 127.71,126.64, 126.56, 109.26, 109.23, 73.62, 72.69, 69.32, 68.53, 66.32,65.98, 25.46, 25.38, 21.48. DART-HRMS: m/z calcd. for C₁₈H₁₈Cl₂O₅S[MH]⁺, 417.0330; Found: 417.0306. IR (solid) vmax: 2988, 2941, 2889,1586, 1556, 1464, 1364, 1188, 1174, 1095, 1035, 979, 809, 662, 552.

4-(4-(4-(4-((2-((1H-1,2,4-triazol-1-yl)methyl)-2-(2,4-dichlorophenyl)-1,3-dioxolan-4-yl)methoxy)phenyl)piperazin-1-yl)phenyl)-2-(sec-butyl)-2,4-dihydro-3H-1,2,4-triazol-3-one(1a). To a solution of 45a (50 mg, 0.127 mol) in DMSO (2 mL) was addeddioxolane tosylate 59a (67 mg, 0.139 mmol) followed by Cs₂CO₃ (0.41 mg,1.27 mmol). The mixture was warmed to 80° C. and stirred for 16 h. Themixture was cooled to RT and water was added slowly (6 mL) with vigorousstirring to form a precipitate. The precipitate was filtered, washedwith water, and determined to be the product with only DMOS as animpurity. The precipitate was dissolve in EtOAc (60 mL) and washed withwater (50 mL). The aqueous layer was washed with EtOAc (1×50 mL) and thecombined organic layers were dried (Na₂SO₄), filtered, and concentrated.The crude residue was purified by column chromatography (SiO₂, 0-80%acetone in hexanes) and sonicated in pentanes to produce 1a as a whitesolid (62 mg, 69%). ¹H NMR (500 MHz, CDCl₃) δ 8.20 (d, J=8.2 Hz, 1H),7.91 (d, J=10.6 Hz, 1H), 7.64-7.55 (m, 2H), 7.50-7.39 (m, 3H), 7.06-6.99(m, 2H), 6.94 (d, J=9.1 Hz, 1H), 6.88 (d, J=8.9 Hz, 1H), 6.84-6.77 (m,1H), 6.69-6.61 (m, 1H), 4.88-4.70 (m, 2H), 4.40-4.19 (m, 2H), 3.96-3.87(m, 1H), 3.85-3.76 (m, 2H), 3.54-3.44 (m, 1H), 3.36 (m, 4H), 3.23 (m,4H), 1.87 (m, 1H), 1.72 (m, 1H), 1.39 (dd, J=6.7, 1.4 Hz, 3H), 0.91 (m,3H). ¹³C NMR (126 MHz, CDCl₃) δ 152.52, 152.43, 151.94, 151.49, 151.31,150.45, 145.94, 145.89, 144.81, 144.60, 135.98, 135.70, 134.97, 133.97,133.81, 133.04, 132.89, 131.35, 131.06, 129.52, 129.42, 127.15, 126.98,125.87, 123.46, 118.37, 118.27, 116.58, 115.19, 115.08, 74.62, 67.57,67.37, 54.30, 53.53, 52.58, 50.49, 49.15, 28.35, 19.17, 10.70.DART-HRMS: m/z calcd. for C₃₅H₃₈Cl₂N₈O₄ [MH]⁺, 705.2471; Found:705.2474. IR (solid) vmax: 3125, 2966, 2832, 1698, 1585, 1551, 1510,1450, 1379, 1228, 1184, 1139, 1042, 976, 944, 824, 736. Purity: 98.0%(Method A).

1-(sec-butyl)-4-(4-(4-(4-((2-(2,4-dichlorophenyl)-2-methyl-1,3-dioxolan-4-yl)methoxy)phenyl)piperazin-1-yl)phenyl)-1H-1,2,4-triazol-5(4H)-one(2a). To a solution of 45a (64 mg, 0.163 mmol) in DMSO (10 mL) was addedsodium hydride (1.14 mmol). aThe mixture was warmed to 50° C. andstirred for 2 h. To this solution was added 61a (62 mg, 0.148 mmol inDMSO, 5 mL). The solution was warmed to 90° C. and stirred for 12 h. Themixture was cooled to RT and H₂O (30 mL) was added slowly with vigorousstirring. The mixture was washed with EtOAc (3×100 mL), and the organiclayers were combined, dried (Na₂SO₄), filtered, and concentrated. Thecrude residue was purified by column chromatography (SiO₂, 0-5% MeOH inDCM) to afford 2a as a reddish-brown solid in modest yield (34%). Aportion of 2a was dissolved in chloroform and slow evaporation providedoff-white crystals that were utilized for the biological assays. ¹H NMR(500 MHz, CDCl₃) δ 7.66-7.59 (m, 2H), 7.46-7.36 (m, 3H), 7.21 (m, 1H),7.06-7.00 (m, 2H), 7.01-6.71 (m, 4H), 4.37-4.25 (m, 2H), 4.12 (m, 1H),4.01 (m, 1H), 3.96 (m, 1H), 3.79 (m, 1H), 3.36 (d, J=6.2 Hz, 4H),3.31-3.17 (m, 4H), 1.89-1.82 (m, 1H), 1.82 (s, 3H), 1.78 (s, 1H), 1.72(m, 1H), 1.39 (d, J=6.7 Hz, 3H), 0.90 (t, J=7.4 Hz, 3H). ¹³C NMR (126MHz, CDCl₃) δ 152.0, 150.5, 138.1, 134.5, 133.8, 132.8, 131.1, 128.8,128.5, 126.7, 126.6, 125.9, 123.5, 118.4, 116.6, 115.5, 109.0, 73.9,69.2, 66.9, 52.7, 50.6, 49.2, 28.4, 25.8, 25.7, 19.2, 10.7. DART-HRMS:m/z calcd. for C₃₃H₃₈Cl₂N₅O₄ [MH]⁺, 638.2301; Found: 638.2298. IR(solid) vmax 2960, 2922, 2874, 2850, 1696, 1552, 1509, 1462, 1449, 1376,1226, 1192, 1149, 1076, 1034, 870, 734. Purity: 98.0% (Method A).

4-(4-(4-(4-((2-((1H-1,2,4-triazol-1-yl)methyl)-2-phenyl-1,3-dioxolan-4-yl)methoxy)phenyl)piperazin-1-yl)phenyl)-1-(sec-butyl)-1H-1,2,4-triazol-5(4H)-one(3a). ITZ analogue 3a was prepared using the general method describedabove for analogue 1a utilizing the requisite linker/side chain anddioxolane intermediates (23 mg, 47%). ¹H NMR (500 MHz, CDCl₃) δ 8.20 (s,1H), 7.91 (s, 1H), 7.61 (s, 1H), 7.57-7.51 (m, 2H), 7.46-7.34 (m, 5H),7.06-7.00 (m, 2H), 6.96-6.90 (m, 2H), 6.82-6.76 (m, 2H), 4.54 (d, J=1.6Hz, 2H), 4.39-4.25 (m, 2H), 3.90 (dd, J=8.4, 6.7 Hz, 1H), 3.77 (m, 2H),3.44 (m, 1H), 3.39-3.33 (m, 4H), 3.26-3.16 (m, 4H), 1.93-1.80 (m, 1H),1.78-1.65 (m, 1H), 1.39 (d, J=6.8 Hz, 3H), 0.90 (t, J=7.4 Hz, 3H). ¹³CNMR (126 MHz, CDCl₃) δ 152.45, 151.61, 145.20, 139.21, 134.33, 129.63,129.08, 126.23, 123.97, 118.89, 117.09, 115.69, 74.96, 68.39, 67.73,56.33, 53.09, 51.03, 49.65, 28.86, 19.67, 11.21. DART-HRMS: m/z calcd.for C₃₅H₄₀N₈O₄ [MH]⁺, 637.3251; Found: 637.3271. IR (solid) vmax 3122,3058, 2961, 2825, 1693, 1602, 1551, 1508, 1448, 1388, 1327, 1296, 1226,1180, 1135, 1939, 944, 823, 736, 701, 676. Purity: 97.1% (Method A).

4-(4-(4-(4-((2-((1H-1,2,4-triazol-1-yl)methyl)-2-methyl-1,3-dioxolan-4-yl)methoxy)phenyl)piperazin-1-yl)phenyl)-2-(sec-butyl)-2,4-dihydro-3H-1,2,4-triazol-3-one(4a). ITZ analogue 4a was prepared using the general method describedabove for analogue 1a utilizing the requisite linker/side chain anddioxolane intermediates. (45%) ¹H NMR (500 MHz, CDCl₃) δ 8.30-8.10 (m,1H), 7.94 (s, 1H), 7.62 (s, 1H), 7.43 (d, J=8.5 Hz, 2H), 7.03 (d, J=8.9Hz, 2H), 6.97-6.87 (m, 2H), 6.84 (t, J=9.9 Hz, 2H), 4.49 (t, J=5.8 Hz,1H), 4.39 (s, 1H), 4.36-4.24 (m, 2H), 4.15 (m, 1H), 4.03-3.84 (m, 1H),3.79 (m, 1H), 3.69-3.59 (m, 1H), 3.42-3.33 (m, 4H), 3.24 (t, J=5.0 Hz,4H), 1.92-1.81 (m, 1H), 1.72 (m, 1H), 1.44 (s, 1H), 1.43-1.36 (m, 5H),0.90 (t, J=7.4 Hz, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 152.7, 152.0, 151.4,150.5, 145.9, 144.6, 133.9, 125.9, 123.5, 118.5, 118.4, 116.6, 115.3,115.2, 108.0, 75.4, 74.8, 68.5, 67.7, 67.5, 67.1, 55.7, 55.6, 52.6,50.6, 49.2, 28.4, 23.5, 22.5, 19.2, 10.7. IR (solid) vmax 3121, 3053,2930, 2850, 2809, 1702, 1683, 1548, 1510, 1471, 1452, 1336, 1251, 1134,1106, 1068, 1050, 940, 883, 735. DART-HRMS: m/z calcd. for C₃₀H₃₉N₈O₄[MH]⁺, 575.3094; Found: 575.3090. IR (solid) vmax: 2967, 2934, 2878,2837, 1701, 1554, 1510, 1450, 1382, 1225, 1181, 1136, 1042, 1017, 942,826, 784. Purity: 95.1% (Method A).

1-(sec-butyl)-4-(4-(4-(4-((2,2-dimethyl-1,3-dioxolan-4-yl)methoxy)phenyl)piperazin-1-yl)phenyl)-1H-1,2,4-triazol-5(4H)-one(5a). ITZ analogue 5a was prepared using the general method describedabove for analogue 2a utilizing the requisite linker/side chain anddioxolane intermediates. The crude residue was purified via columnchromatography (SiO₂, 0-5% MeOH in DCM) to afford 5a in modest yield (14mg, 21%) NMR (500 MHz, CDCl₃) δ 7.61 (d, J=7.2 Hz, 1H), 7.43 (d, J=8.4Hz, 2H), 7.03 (d, J=8.5 Hz, 2H), 6.94 (s, 1H), 6.89 (d, J=8.4 Hz, 2H),4.46 (m, 1H), 4.29 (m, 1H), 4.16 (t, J=7.4 Hz, 1H), 4.04 (m, 1H), 3.90(m, 2H), 3.37 (s, 2H), 3.24 (s, 2H), 1.86 (m, 1H), 1.71 (m, 1H), 1.46(s, 3H), 1.39 (d, J=7.8 Hz, 6H), 0.91 (t, J=7.4 Hz, 3H). ¹³C NMR (126MHz, CDCl₃) δ 152.44, 134.28, 124.08, 123.96, 118.93, 117.14, 115.81,110.13, 74.49, 69.77, 67.32, 53.11, 51.11, 49.62, 33.08, 28.86, 27.22,25.79, 19.66, 11.20. DART-HRMS: m/z calcd. for C₂₈H₃₇N₅O₄ [MH]⁺,508.2924; Found: 508.2909. IR (solid) vmax: 3126, 3060, 2967, 2926,2878, 2828, 2212, 1681, 1584, 1556, 1510, 1452, 1380, 1226, 1149, 1037,941, 819, 736. Purity: 97.5% (Method B).

4-(4-(4-(4-((2-((1H-1,2,4-triazol-1-yl)methyl)-2-(2,4-dichlorophenyl)-1,3-dioxolan-4-yl)methoxy)phenyl)piperazin-1-yl)phenyl)-2-((S)-sec-butyl)-2,4-dihydro-3H-1,2,4-triazol-3-one(6a). ITZ analogue 6a was prepared using the general method describedabove for analogue 1a utilizing the requisite linker/side chain anddioxolane intermediates (55%). ¹H NMR (500 MHz, CDCl₃) δ 8.20 (s, 1H),7.89 (s, 1H), 7.61 (s, 1H), 7.57 (d, J=8.3 Hz, 1H), 7.47 (d, J=2.1 Hz,1H), 7.43 (d, J=8.9 Hz, 2H), 7.03 (d, J=9.0 Hz, 2H), 6.94 (m, 2H), 6.80(m, 2H), 4.80 (m, 2H), 4.36 (m, 1H), 4.28 (m, 1H), 3.92 (m, 1H), 3.81(m, 2H), 3.48 (m, 1H), 3.36 (m, 4H), 3.23 (m, 4H), 1.86 (m, 1H), 1.72(m, 1H), 1.39 (d, J=6.7 Hz, 3H), 0.90 (t, J=7.4 Hz, 3H). ¹³C NMR (126MHz, CDCl₃) δ 152.6, 151.4, 150.5, 146.0, 144.8, 136.0, 134.0, 133.8,133.1, 131.4, 129.6, 127.2, 125.9, 123.5, 118.4, 116.6, 115.2, 109.9,107.6, 74.7, 67.6, 67.4, 53.6, 52.6, 50.5, 49.2, 28.4, 19.2, 10.7.DART-HRMS: m/z calcd. for C₃₅H₃₉Cl₂N₈O₄ [MH]⁺, 705.2471; Found:705.2465. IR (solid) vmax: 2967, 2930, 2878, 1695, 1586, 1552, 1509,1451, 1378, 1226, 1183, 1130, 1038, 947, 816, 736. Purity: 97.9% (MethodA).

4-(4-(4-(4-((2-((1H-1,2,4-triazol-1-yl)methyl)-2-(2,4-dichlorophenyl)-1,3-dioxolan-4-yl)methoxy)phenyl)piperazin-1-yl)phenyl)-2-((R)-sec-butyl)-2,4-dihydro-3H-1,2,4-triazol-3-one(7a). ITZ analogue 7a was prepared using the general method describedabove for analogue 1a utilizing the requisite linker/side chain anddioxolane intermediates (77%). ¹H NMR (500 MHz, CDCl₃) δ 8.20 (s, 1H),7.89 (s, 1H), 7.64-7.54 (m, 2H), 7.50-7.40 (m, 3H), 7.03 (d, J=8.6 Hz,2H), 6.95 (s, 1H), 6.81 (d, J=8.4 Hz, 2H), 4.84 (d, J=14.7 Hz, 1H), 4.76(d, J=14.7 Hz, 1H), 4.39-4.25 (m, 2H), 3.92 (m, 1H), 3.86-3.77 (m, 2H),3.50 (m, 1H), 3.38 (s, 2H), 3.25 (s, 3H), 1.87 (m, 1H), 1.72 (m, 1H),1.39 (d, J=6.7 Hz, 3H), 0.91 (t, J=7.4 Hz, 3H). ¹³C NMR (126 MHz, CDCl₃)δ 152.5, 152.0, 151.3, 150.5, 146.0, 136.0, 134.0, 133.8, 133.1, 131.4,129.5, 127.2, 125.9, 123.5, 118.4, 116.6, 115.2, 107.6, 77.2, 74.7,67.6, 67.4, 53.6, 52.6, 50.5, 49.2, 28.4, 19.2, 10.7. DART-HRMS: m/zcalcd. for C₃₅H₃₉Cl₂N₈O₄ [MH]⁺, 705.2471; Found: 705.2468 IR (solid)vmax: 3067, 2966, 2934, 2878, 2832, 1695, 1585, 1551, 1509, 1450, 1379,1225, 1180, 1136, 1039, 944, 820, 794. Purity: 95.0% (Method A).

4-(4-(4-(4-((2-((1H-1,2,4-triazol-1-yl)methyl)-2-(2,4-dichlorophenyl)-1,3-dioxolan-4-yl)methoxy)phenyl)piperazin-1-yl)phenyl)-1-propyl-1H-1,2,4-triazol-5(4H)-one(8a). ITZ analogue 8a was prepared using the general method describedabove for analogue 2a utilizing the requisite linker/side chain anddioxolane intermediates (50 mg, 68%). NMR (500 MHz, CDCl₃) δ 8.20 (s,1H), 7.89 (s, 1H), 7.59 (d, J=21.6 Hz, 2H), 7.48 (s, 1H), 7.41 (s, 2H),7.04 (s, 2H), 6.93 (s, 2H), 6.81 (s, 2H), 4.80 (d, J=23.3 Hz, 2H), 4.36(s, 1H), 3.92 (s, 1H), 3.82 (s, 4H), 3.52 (s, 1H), 3.37 (s, 4H), 3.24(s, 5H), 1.84 (s, 2H), 0.98 (s, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 152.60,136.48, 134.30, 133.55, 131.85, 130.02, 127.65, 123.98, 117.08, 115.73,108.05, 75.13, 68.11, 54.05, 51.01, 49.60, 47.62, 22.43, 11.50.DART-HRMS: m/z calcd. for C₃₄H₃₆Cl₂N₈O₄ [MH]⁺, 691.2315; Found:691.2329. IR (solid) vmax: 3068, 2960, 2925, 2873, 2835, 1696, 1585,1553, 1510, 1452, 1379, 1225, 1160, 1136, 1045, 944, 823, 794. Purity:95.2% (Method A).

1-(4-((2-((1H-1,2,4-triazol-1-yl)methyl)-2-(2,4-dichlorophenyl)-1,3-dioxolan-4-yl)methoxy)phenyl)-4-(4-nitrophenyl)piperazine(10a). To a solution of 68a (100 mg, 0.336 mmol) in DMSO (4 mL) wasadded Cs₂CO₃ (1.1 g, 3.36 mmol) and 59a (0.29 g, 0.604 mmol). Thesolution was warmed to 90° C. and stirred for 12 h. The mixture wascooled to room temperature and water was added slowly with vigorousstirring (˜6 mL). A yellow precipitate formed, which was filtered andrecrystallized in EtOH to yield 10a (150 mg, 73%). a¹H NMR (500 MHz,CDCl₃) δ 8.22-8.12 (m, 3H), 7.90 (s, 1H), 7.58 (d, J=8.5 Hz, 1H), 7.48(d, J=2.1 Hz, 1H), 6.95-6.86 (m, 4H), 6.86-6.78 (m, 2H), 4.84 (d, J=14.8Hz, 1H), 4.76 (d, J=14.7 Hz, 1H), 4.36 (m, 1H), 3.92 (m, 1H), 3.85-3.75(m, 3H), 3.61-3.55 (m, 4H), 3.48 (m, 1H), 3.26-3.20 (m, 4H). ¹³C NMR(126 MHz, CDCl₃) δ 154.79, 152.90, 151.42, 145.67, 144.94, 138.79,136.14, 134.08, 133.17, 131.49, 129.63, 127.28, 125.99, 118.58, 115.39,112.90, 107.69, 74.72, 67.71, 67.45, 53.63, 50.33, 47.29. DART-HRMS: m/zcalcd. for C₂₉H₂₈Cl₂N₆O₅ [MH]⁺, 611.1577; Found: 611.1601. IR (solid)vmax: 3116, 2923, 2852, 1589, 1557, 1506, 1456, 1377, 1318, 1226, 1136,1029, 975, 942, 896, 823, 737, 691. Purity: 95.0% (Method A).

4-(4-(4-((2-((1H-1,2,4-triazol-1-yl)methyl)-2-(2,4-dichlorophenyl)-1,3-dioxolan-4-yl)methoxy)phenyl)piperazin-1-yl)aniline(11a). 10% palladium on carbon (1.04 mg, 5% mole ratio) was added to adry round bottom flask. Ethanol (25 mL) was added followed by slowaddition of 10a (120 mg, 0.196 mmol). Hydrazine monohydrate (0.06 mL,1.96 mmol) was added dropwise and the mixture was stirred at reflux for2 h. Upon cooling to RT, the mixture was filtered through celite. Thecelite was washed with ethanol (100 mL) and chloroform (250 mL) toensure complete elution of the aniline. The filtrate was concentrated toafford a yellow solid, which was recrystallized in EtOH to afford 11a(70 mg, 61%). ¹H NMR (500 MHz, CDCl₃) δ 8.20 (s, 1H), 8.15 (d, J=9.4 Hz,2H), 7.89 (s, 1H), 7.57 (d, J=8.4 Hz, 1H), 7.47 (m, 1H), 7.25 (m, 1H),6.92 (m, 2H), 6.88 (d, J=9.5 Hz, 2H), 6.80 (d, J=9.0 Hz, 2H), 4.80 (m,2H), 4.36 (m, 1H), 3.91 (m, 1H), 3.80 (m, 2H), 3.58 (m, 4H), 3.47 (m,1H), 3.22 (m, 4H). ¹³C NMR (126 MHz,) δ 154.7, 152.8, 151.3, 145.6,144.9, 138.7, 136.1, 134.0, 133.1, 131.4, 129.6, 127.2, 125.9, 118.5,115.3, 112.8, 107.6, 74.6, 67.6, 67.4, 53.6, 50.3, 47.2. DART-HRMS: m/zcalcd. for C₂₉H₃₁Cl₂N₆O₃ [MH]⁺, 581.1835; Found: 581.1818. IR (solid)vmax: 3084, 2886, 2827, 1558, 1504, 1313, 1224, 113, 1030, 942, 821,749. Purity: 96.1% (Method A).

Second Generation ITZ Intermediates and Final Analogues (12a-25a).

(2-(2,4-dichlorophenyl)-2-methyl-1,3-dioxolan-4-yl)methyl4-methylbenzenesulfonate (61a′) and(2-(2,4-dichlorophenyl)-2-methyl-1,3-dioxolan-4-yl)methyl4-methylbenzenesulfonate (61a″). The defined trans (61a′) and cis (61a″)mixtures of des-triazole dioxolane tosylates were prepared by extensivecolumn chromatography (SiO₂, 0-20% EtOAc in Hex) on the complete mixtureof tosylate stereoisomers 61a. Fraction 1 (R_(f)˜0.7 in 3:1 Hex:EtOAc)was characterized as 2,4-anti-substituted dioxolanes (61a′) and Fraction2 (R_(f)˜0.6 in 3:1 Hex:EtOAc) was characterized as the2,4-syn-substituted dioxolanes (61a″) [Combined yield=90%; Fraction 1(61a′)=60%; Fraction 2 (61a″)=35%]. Fraction 1 crystallized over timewhereas Fraction 2 remained a clear oil.

61a′. ¹H NMR (500 MHz, CDCl₃) δ 7.82 (d, J=8.2 Hz, 2H), 7.49 (d, J=8.3Hz, 1H), 7.39-7.32 (m, 3H), 7.20 (m, 1H), 4.21-4.14 (m, 1H), 4.10 (m,1H), 4.03 (m, 1H), 3.87 (m, 1H), 3.72 (m, 1H), 2.46 (s, 3H), 1.70 (s,3H). ¹³C NMR (126 MHz, CDCl₃) δ 145.11, 137.59, 134.67, 132.69, 132.66,131.17, 129.92, 128.67, 128.00, 126.78, 109.43, 72.80, 69.36, 66.22,25.55, 21.65. DART-HRMS: m/z calcd. for C₁₈H₁₈Cl₂O₅S [MH]⁺, 417.0330;Found: 417.0345. IR (solid) vmax: 3007, 2989, 2937, 2894, 1585, 1557,1465, 1359, 1186, 1172, 1093, 1036, 811, 751, 663, 552, 492.

61a″. ¹H NMR (500 MHz, CDCl₃) δ 7.76-7.61 (m, 2H), 7.46 (d, J=8.5 Hz,1H), 7.36-7.29 (m, 3H), 7.11 (m, 1H), 4.52-4.38 (m, 1H), 4.20 (m, 1H),3.92 (m, 1H), 3.80 (m, 1H), 3.57 (m, 1H), 2.47 (s, 3H), 1.70 (d, J=1.1Hz, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 145.10, 138.65, 134.45, 132.59,132.38, 130.92, 129.87, 128.21, 127.90, 126.72, 109.48, 73.73, 68.57,66.60, 25.63, 21.67. DART-HRMS: m/z calcd. for C₁₈H₁₈Cl₂O₅S [MH]⁺,417.0330; Found: 417.0347. IR (solid) vmax: 2988, 2939, 2888, 1586,1556, 1464, 1364, 1188, 1174, 1095, 1034, 979, 808, 662, 522.

((2R,4S)-2-(2,4-dichlorophenyl)-2-methyl-1,3-dioxolan-4-yl)methyl4-methylbenzenesulfonate (64) and((2S,4S)-2-(2,4-dichlorophenyl)-2-methyl-1,3-dioxolan-4-yl)methyl4-methylbenzenesulfonate (65a). Stereochemically-defined tosylateddes-triazole intermediates 64a and 65a were prepared from 60a andtosylated glycerol 62a using the general method described above for 61a.Following initial column chromatography, ˜740 mg of the 64a:65a mixturewas loaded on a preparative TLC plate (Analtech Uniplate, 20×20 cm, 2000mm coating thickness, Silica G). The plate was developed repeatedly (8×in 8:1 Hex:EtOAc). Following development and separation, the two bandswere stripped from the TLC plate and the compounds removed from thesilica beads by gentle stirring (5% MeOH in DCM, 200 mL, 12 h).

(64a). White solid, 45%. R_(f)=0.7 in 3:1 Hex:EtOAc. ¹H NMR (500 MHz,CHCl₃) 7.78 (m, 3H), 7.48 (d, J=8.4 Hz, 1H), 7.34 (m, 3H), 7.17 (m, 1H),4.18 (m, 1H), 4.08 (m, 1H), 4.02 (m, 1H), 3.84 (m, 1H), 3.69 (m, 1H),2.43 (s, 3H), 1.67 (s, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 145.0, 143.7,138.9, 137.5, 134.5, 132.5, 132.5, 131.0, 129.8, 129.7, 129.7, 128.6,127.8, 127.3, 126.6, 109.2, 72.7, 69.3, 66.0, 25.4, 21.5, 21.3.DART-HRMS: m/z calcd. for C₁₈H₁₈Cl₂O₅S [MH]⁺, 417.0330; Found: 417.0327.IR (solid) vmax: 3093, 3007, 2989, 2916, 2894, 1585, 1556, 1359, 1186,1172, 1093, 1035, 960, 940, 861, 751, 662, 551, 492.

(65a). Clear oil, 25%. R_(f)=0.6 in 3:1 Hex:EtOAc). ¹H NMR (500 MHz,CHCl₃) 7.69 (d, J=8.2 Hz, 2H), 7.46 (d, J=8.4 Hz, 1H), 7.31 (m, 3H),7.10 (m, 1H), 4.44 (m, 1H), 4.19 (m, 1H), 3.91 (m, 1H), 3.80 (m, 1H),3.56 (m, 1H), 2.46 (s, 3H), 1.70 (s, 3H). ¹³C NMR (126 MHz, CDCl₃) δ145.0, 138.6, 134.4, 132.5, 132.3, 130.9, 129.8, 128.2, 127.8, 126.7,109.4, 73.7, 68.5, 66.5, 29.6, 25.6, 21.6. DART-HRMS: m/z calcd. forC₁₈H₁₈Cl₂O₅S [MH]⁺, 417.0330; Found: 417.0325. IR (solid) vmax: 3117,3054, 2955, 2822, 1687, 1551, 1353, 1229, 1187, 1173, 1095, 1033, 967,943, 824, 809, 663, 552, 531.

((2S,4R)-2-(2,4-dichlorophenyl)-2-methyl-1,3-dioxolan-4-yl)methyl4-methylbenzenesulfonate (66) and((2R,4R)-2-(2,4-dichlorophenyl)-2-methyl-1,3-dioxolan-4-yl)methyl4-methylbenzenesulfonate (67a). Stereochemically-defined tosylateddes-triazole intermediates 66a and 67a were prepared from 60a andtosylated glycerol 63a using the general method described above for 61aand purified as described for 64a and 65a.

(66a). Crystalline, 55%. R_(f)=0.6 in 3:1 Hex:EtOAc). ¹H NMR (500 MHz,CHCl₃) 7.81 (m, 3H), 7.49 (d, J=8.4 Hz, 1H), 7.36 (m, 3H), 7.19 (m, 1H),4.18 (m, 1H), 4.09 (m, 1H), 4.02 (m, 1H), 3.86 (m, 1H), 3.70 (m, 1H),2.45 (s, 3H), 1.69 (s, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 145.1, 137.5,134.6, 132.6, 132.6, 131.1, 129.9, 128.6, 127.9, 127.9, 126.7, 109.4,72.7, 69.3, 66.1, 25.5, 21.6. DART-HRMS: m/z calcd. for C₁₈H₁₈Cl₂O₅S[MH]⁺, 417.0330; Found: 417.0358. IR (solid) vmax: 3094, 3008, 2989,2957, 2936, 2917, 2894, 1593, 1557, 1359, 1186, 1172, 1094, 1036, 961,940, 862, 752, 662, 552, 493.

(67a). Clear oil, 30%. R_(f)=0.5 in 3:1 Hex:EtOAc. ¹H NMR (500 MHz,CHCl₃) 7.69 (m, 2H), 7.46 (d, J=8.4 Hz, 1H), 7.31 (m, 3H), 7.10 (m, 1H),4.45 (m, 1H), 4.20 (m, 1H), 3.91 (m, 1H), 3.80 (m, 1H), 3.56 (m, 1H),2.46 (s, 3H), 1.70 (s, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 145.1, 138.6,134.4, 132.5, 132.3, 130.9, 129.8, 128.2, 127.9, 126.7, 109.4, 73.7,68.5, 66.6, 25.6, 21.6. DART-HRMS: m/z calcd. for C₁₈H₁₈Cl₂O₅S [MH]⁺,417.0330; Found: 417.0356. IR (solid) vmax: 3094, 3007, 2989, 2957,2917, 2894, 1585, 1557, 1358, 1185, 1172, 1093, 1036, 961, 939, 862,751, 662, 551, 492.

General protocol for tosylate/phenol coupling and final analoguepurification. To a solution of alkyl-substituted phenol (43a-46a) (40mg, 0.102 mmol) in DMSO (2.0 mL) was added des-triazole-tosylate(61a′-61a″, 64a-67a) (46 mg, 0.110 mmol) followed by Cs₂CO₃ (0.82 mmol).The mixture was warmed to 80° C. and stirred for 16 h. The mixture wasthen cooled to RT and water was added slowly (6 mL) with vigorousstirring, which resulted in formation of a precipitate. The mixture wastransferred to a separatory funnel, diluted with EtOAc (60 mL) andwashed with water (50 mL). The aqueous layer was washed with EtOAc (1×60mL). The organic layers were combined, dried (MgSO₄), filtered, andconcentrated. The crude residue was purified by column chromatography(SiO₂, 0 to 24% acetone in hexanes) to afford 2a, 12a-25a as white toslightly off-white solids in good yields (45-88%). Final analogues weresubsequently sonicated in pentanes (10-30 min) to remove a “grease-like”impurity (observed in ¹H NMRs at 0.88, 1.31 ppm) and collected forpurity analysis and biological evaluation following filtration on afine-fritted glass Buchner style filter funnel.

4-(4-(4-(4-((2-((1H-1,2,4-triazol-1-yl)methyl)-2-(2,4-dichlorophenyl)-1,3-dioxolan-4-yl)methoxy)phenyl)piperazin-1-yl)phenyl)-2-(sec-butyl)-2,4-dihydro-3H-1,2,4-triazol-3-one(12a). ¹H NMR (500 MHz, CDCl₃) δ 7.62 (m, 2H), 7.42 (m, 3H), 7.23 (m,1H), 7.03 (m, 2H), 6.92 (m, 2H), 4.31 (m, 2H), 4.11 (m, 1H), 4.01 (m,1H), 3.97 (m, 1H), 3.84 (m, 1H), 3.36 (m, 4H), 3.24 (m, 4H), 1.87 (m,1H), 1.81 (s, 3H), 1.72 (m, 1H), 1.39 (d, J=6.7 Hz, 3H), 0.91 (t, J=7.4Hz, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 152.7, 152.0, 150.5, 145.8, 139.2,134.2, 133.8, 132.7, 130.9, 128.5, 126.6, 125.9, 123.5, 118.3, 116.6,115.2, 109.1, 73.9, 69.3, 66.9, 52.6, 50.6, 49.2, 28.4, 25.6, 19.2,10.7. DART-HRMS: m/z calcd. for C₃₃H₃₈Cl₂N₅O₄ [MH]⁺, 638.2301; Found:638.2328. IR (solid) vmax 2961, 2918, 2849, 1694, 1584, 1556, 1509,1449, 1374, 1329, 1294, 1224, 1186, 1149, 1094, 1035, 1017, 942, 873,821, 802, 734. Purity: 97.0% (Method B).

4-(4-(4-(4-((2-((1H-1,2,4-triazol-1-yl)methyl)-2-(2,4-dichlorophenyl)-1,3-dioxolan-4-yl)methoxy)phenyl)piperazin-1-yl)phenyl)-2-(sec-butyl)-2,4-dihydro-3H-1,2,4-triazol-3-one(13a). ¹H NMR (500 MHz, CDCl₃) δ 7.63 (d, J=8.5 Hz, 1H), 7.61 (s, 1H),7.42 (m, 2H), 7.38 (d, J=2.1 Hz, 1H), 7.19 (m, 1H), 7.01 (m, 2H), 6.89(m, 2H), 6.73 (m, 2H), 4.60 (m, 1H), 4.29 (m, 2H), 3.94 (m, 1H), 3.73(m, 1H), 3.35 (m, 4H), 3.21 (m, 4H), 1.85 (m, 1H), 1.77 (s, 3H), 1.71(m, 1H), 1.39 (d, J=6.7 Hz, 3H), 0.90 (t, J=7.4 Hz, 3H). ¹³C NMR (126MHz, CDCl₃) δ 152.7, 152.0, 150.5, 145.8, 139.2, 134.2, 133.8, 132.7,130.9, 128.5, 126.6, 125.9, 123.5, 118.3, 116.6, 115.2, 109.1, 75.0,68.3, 67.3, 52.6, 50.5, 49.2, 28.4, 25.8, 19.2, 10.7. HRMS: m/z calcd.for C₃₃H₃₈Cl₂N₅O₄ [MH]⁺, 638.2301; Found: 638.2325. IR (solid) vmax3102, 3008, 2961, 2918, 2899, 2849, 1699, 1597, 1538, 1515, 1411, 1355,1337, 1254, 1236, 1189 1159, 1106, 1064, 1036, 1024, 999, 936, 896, 831,807, 741. Purity: 95.1% (Method B).

2-((S)-sec-butyl)-4-(4-(4-(4-(((2S,4S)-2-(2,4-dichlorophenyl)-2-methyl-1,3-dioxolan-4-yl)methoxy)phenyl)piperazin-1-yl)phenyl)-2,4-dihydro-3H-1,2,4-triazol-3-one(14a). ¹H NMR (500 MHz, CDCl₃) δ 7.61 (m, 3H), 7.42 (m, 2H), 7.40 (m,1H), 7.23 (m, 1H), 7.02 (m, 2H), 6.94 (m, 2H), 6.88 (m, 2H), 4.31 (m,2H), 4.11 (m, 1H), 4.00 (m, 1H), 3.96 (m, 1H), 3.36 (m, 4H), 3.23 (m,4H), 1.85 (m, 1H), 1.81 (s, 3H), 1.72 (m, 1H), 1.39 (d, J=6.7 Hz, 3H),0.91 (t, J=7.4 Hz, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 152.9, 152.0, 150.5,145.9, 138.1, 134.5, 133.8, 132.8, 131.1, 128.8, 126.7, 125.9, 123.5,118.4, 116.6, 115.4, 109.0, 73.9, 69.2, 66.9, 52.6, 50.6, 49.2, 28.4,25.7, 19.2, 10.7. DART-HRMS: m/z calcd. for C₃₃H₃₈Cl₂N₅O₄ [MH]⁺,638.2301; Found: 638.2282. IR (solid) vmax 2962, 2875, 2826, 1694, 1555,1509, 1448, 1374, 1224, 1186, 1149, 1035, 942, 824, 735. Purity: 97.4%(Method A).

2-((S)-sec-butyl)-4-(4-(4-(4-(((2R,4S)-2-(2,4-dichlorophenyl)-2-methyl-1,3-dioxolan-4-yl)methoxy)phenyl)piperazin-1-yl)phenyl)-2,4-dihydro-3H-1,2,4-triazol-3-one(15a). ¹H NMR (500 MHz, CDCl₃) δ 7.64 (d, J=8.4 Hz, 2H), 7.61 (s, 1H),7.42 (d, J=8.5 Hz, 2H), 7.38 (d, J=2.2 Hz, 2H), 7.19 (m, 1H), 7.02 (d,J=8.5 Hz, 2H), 6.90 (d, J=8.5 Hz, 2H), 6.73 (d, J=8.5 Hz, 2H), 4.60 (m,1H), 4.30 (m, 2H), 3.94 (m, 1H), 3.73 (m, 2H), 3.35 (m, 4H), 3.21 (m,4H), 1.86 (m, 1H), 1.78 (s, 3H), 1.72 (m, 1H), 1.39 (d, J=6.7 Hz, 3H),0.91 (t, J=7.4 Hz, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 152.8, 152.0, 150.5,145.8, 139.3, 134.3, 133.8, 132.7, 130.9, 128.5, 126.6, 125.9, 123.5,118.4, 116.6, 115.2, 109.1, 75.0, 68.4, 67.3, 52.6, 50.6, 49.2, 28.4,25.8, 19.2, 10.7. DART-HRMS: m/z calcd. for C₃₃H₃₈Cl₂N₅O₄ [MH]⁺,638.2301; Found: 638.2288. IR (solid) vmax 2923, 2851, 1714, 1703, 1683,1613, 1548, 1509, 1452, 1374, 1271, 1226, 1188, 1150, 1094, 1035, 965,942, 817, 735. Purity: 95.5% (Method A).

2-((S)-sec-butyl)-4-(4-(4-(4-(((2R,4R)-2-(2,4-dichlorophenyl)-2-methyl-1,3-dioxolan-4-yl)methoxy)phenyl)piperazin-1-yl)phenyl)-2,4-dihydro-3H-1,2,4-triazol-3-one(16a). ¹H NMR (500 MHz, CDCl₃) δ 7.61 (m, 2H), 7.41 (m, 3H), 7.23 (m,1H), 7.02 (m, 2H), 6.94 (m, 2H), 6.89 (m, 2H), 4.31 (m, 2H), 4.11 (m,1H), 4.00 (m, 1H), 3.96 (m, 1H), 3.84 (m, 1H), 3.36 (m, 4H), 3.23 (m,4H), 1.86 (m, 1H), 1.81 (s, 3H), 1.72 (m, 1H), 1.39 (d, J=6.7 Hz, 3H),0.91 (t, J=7.4 Hz, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 152.9, 152.0, 150.5,145.9, 138.1, 134.5, 133.8, 132.8, 131.1, 128.8, 126.7, 125.9, 123.5,118.4, 116.6, 115.4, 109.0, 73.9, 69.2, 66.9, 52.6, 50.6, 49.2, 28.4,25.7, 19.2, 10.7. DART-HRMS: m/z calcd. for C₃₃H₃₈Cl₂N₅O₄ [MH]⁺,638.2301; Found: 638.2284. IR (solid) vmax 2966, 2935, 2874, 2824, 1693,1584, 1555, 1508, 1464, 1447, 1373, 1293, 1223, 1186, 1149, 1095, 1035,942, 875, 824, 734. Purity: 97.3% (Method A).

2-((S)-sec-butyl)-4-(4-(4-(4-(((2S,4R)-2-(2,4-dichlorophenyl)-2-methyl-1,3-dioxolan-4-yl)methoxy)phenyl)piperazin-1-yl)phenyl)-2,4-dihydro-3H-1,2,4-triazol-3-one(17a). ¹H NMR (500 MHz, CDCl₃) δ 7.64 (d, J=8.5 Hz, 1H), 7.61 (s, 1H),7.42 (m, 2H), 7.38 (d, J=2.1 Hz, 1H), 7.19 (m, 1H), 7.02 (m, 2H), 6.89(m, 2H), 6.73 (m, 2H), 4.60 (m, 1H), 4.30 (m, 2H), 3.94 (m, 1H), 3.73(m, 2H), 3.35 (m, 4H), 3.21 (m, 4H), 1.85 (m, 1H), 1.78 (s, 3H), 1.72(m, 1H), 1.39 (d, J=6.7 Hz, 3H), 0.91 (t, J=7.4 Hz, 3H). ¹³C NMR (126MHz, CDCl₃) δ 152.8, 152.0, 150.5, 145.8, 139.3, 134.3, 133.8, 132.7,130.9, 128.5, 126.6, 125.9, 123.5, 118.4, 118.4, 116.6, 115.2, 109.1,75.0, 68.4, 67.3, 52.6, 50.6, 49.2, 28.4, 25.8, 19.2, 10.7. DART-HRMS:m/z calcd. for C₃₃H₃₈Cl₂N₅O₄ [MH]⁺, 638.2301; Found: 638.2281. IR(solid) vmax 2919, 2876, 2849, 1693, 1585, 1555, 1509, 1450, 1375, 1329,1295, 1225, 1188, 1149, 1095, 1034, 934, 872, 819, 734. Purity: 98.4%(Method A).

2-((R)-sec-butyl)-4-(4-(4-(4-(((2S,4S)-2-(2,4-dichlorophenyl)-2-methyl-1,3-dioxolan-4-yl)methoxy)phenyl)piperazin-1-yl)phenyl)-2,4-dihydro-3H-1,2,4-triazol-3-one(18a). ¹H NMR (500 MHz, CDCl₃) δ 7.61 (m, 2H), 7.42 (m, 2H), 7.41 (m,1H), 7.23 (m, 1H), 7.03 (m, 2H), 6.94 (m, 2H), 6.89 (m, 2H), 4.31 (m,2H), 4.11 (m, 1H), 4.01 (m, 1H), 3.96 (m, 1H), 3.84 (m, 1H), 3.36 (m,4H), 3.23 (m, 4H), 1.87 (m, 1H), 1.81 (s, 3H), 1.72 (m, 1H), 1.39 (d,J=6.7 Hz, 3H), 0.91 (t, J=7.4 Hz, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 152.9,152.0, 150.5, 145.9, 138.1, 134.5, 133.8, 132.8, 131.1, 128.8, 126.7,125.9, 123.5, 118.4, 116.6, 115.4, 109.0, 73.9, 69.2, 66.9, 52.6, 50.6,49.2, 28.4, 25.7, 19.2, 10.7. DART-HRMS: m/z calcd. for C₃₃H₃₈Cl₂N₅O₄[MH]⁺, 638.2301; Found: 638.2282. IR (solid) vmax 2967, 2932, 2878,2827, 1695, 1612, 1586, 1552, 1510, 1460, 1377, 1292, 1253, 1225, 1187,1147, 1096, 1036, 940, 827, 735. Purity: 95.0% (Method B).

2-((R)-sec-butyl)-4-(4-(4-(4-(((2R,4S)-2-(2,4-dichlorophenyl)-2-methyl-1,3-dioxolan-4-yl)methoxy)phenyl)piperazin-1-yl)phenyl)-2,4-dihydro-3H-1,2,4-triazol-3-one(19a). ¹H NMR (500 MHz, CDCl₃) δ 7.64 (d, J=8.5 Hz, 1H), 7.61 (s, 1H),7.42 (m, 2H), 7.38 (m, 1H), 7.20 (m, 1H), 7.02 (m, 2H), 6.90 (m, 2H),6.73 (m, 2H), 4.60 (m, 1H), 4.30 (m, 1H), 3.94 (m, 1H), 3.73 (m, 1H),3.35 (m, 4H), 3.21 (m, 4H), 1.86 (m, 1H), 1.78 (s, 3H), 1.71 (m, 1H),1.39 (d, J=6.7 Hz, 3H), 0.91 (t, J=7.4 Hz, 3H). ¹³C NMR (126 MHz, CDCl₃)δ 152.9, 152.0, 150.5, 145.9, 138.1, 134.5, 133.8, 132.8, 131.1, 128.7,126.7, 125.9, 123.5, 118.4, 116.6, 115.4, 109.0, 73.9, 69.2, 66.9, 52.6,50.6, 49.2, 28.4, 25.7, 19.2, 10.7. DART-HRMS: m/z calcd. forC₃₃H₃₈Cl₂N₅O₄ [MH]⁺, 638.2301; Found: 638.2287. IR (solid) vmax 3060,2967, 2932, 2830, 1698, 1611, 1586, 1550, 1512, 1461, 1384, 1296, 1252,1225, 1189, 1149, 1098, 1036, 940, 896, 824, 735. Purity: 95.0% (MethodB).

2-((R)-sec-butyl)-4-(4-(4-(4-(((2R,4R)-2-(2,4-dichlorophenyl)-2-methyl-1,3-dioxolan-4-yl)methoxy)phenyl)piperazin-1-yl)phenyl)-2,4-dihydro-3H-1,2,4-triazol-3-one(20a). ¹H NMR (500 MHz, CDCl₃) δ 7.61 (m, 2H), 7.43 (m, 2H), 7.41 (m,1H), 7.23 (m, 1H), 7.03 (m, 2H), 6.94 (m, 2H), 6.89 (m, 2H), 4.31 (m,2H), 4.11 (m, 1H), 4.01 (m, 1H), 3.96 (m, 1H), 3.84 (m, 1H), 3.36 (m,4H), 3.23 (m, 4H), 1.86 (m, 1H), 1.81 (s, 3H), 1.72 (m, 1H), 1.39 (d,J=6.7 Hz, 3H), 0.91 (t, J=7.4 Hz, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 153.0,152.0, 150.5, 145.9, 138.1, 134.5, 133.8, 132.8, 131.1, 128.8, 126.7,125.9, 123.5, 118.4, 116.6, 115.4, 109.0, 73.9, 69.2, 67.0, 52.6, 50.6,49.2, 28.4, 25.7, 19.2, 10.7. DART-HRMS: m/z calcd. for C₃₃H₃₈Cl₂N₅O₄[MH]⁺, 638.2301; Found: 638.2285. IR (solid) vmax 2971, 2937, 2879,2827, 1697, 1509, 1451, 1376, 1296, 1225, 1194, 1147, 1073, 1035, 942,819, 734. Purity: 97.5% (Method B).

2-((R)-sec-butyl)-4-(4-(4-(4-(((2S,4R)-2-(2,4-dichlorophenyl)-2-methyl-1,3-dioxolan-4-yl)methoxy)phenyl)piperazin-1-yl)phenyl)-2,4-dihydro-3H-1,2,4-triazol-3-one(21a). ¹H NMR (500 MHz, CDCl₃) δ 7.64 (d, J=8.5 Hz, 2H), 7.61 (s, 1H),7.42 (m, 2H), 7.37 (m, 1H), 7.19 (m, 1H), 7.02 (m, 2H), 6.90 (m, 2H),6.73 (m, 2H), 4.60 (m, 1H), 4.30 (m, 2H), 3.95 (m, 1H), 3.73 (m, 2H),3.35 (m, 4H), 3.22 (m, 4H), 1.86 (m, 1H), 1.78 (s, 3H), 1.72 (m, 1H),1.39 (d, J=6.7 Hz, 3H), 0.91 (t, J=7.4 Hz, 3H). ¹³C NMR (126 MHz, CDCl₃)δ 152.8, 152.0, 150.5, 145.9, 139.3, 134.3, 133.8, 132.7, 130.9, 128.5,126.6, 125.9, 123.5, 118.4, 118.4, 116.6, 115.2, 109.1, 75.0, 68.4,67.3, 52.6, 50.6, 49.2, 28.4, 25.8, 19.2, 10.7. DART-HRMS: m/z calcd.for C₃₃H₃₈Cl₂N₅O₄ [MH]⁺, 638.2301; Found: 638.2284. IR (solid) vmax2966, 2932, 2904, 2829, 1695, 1553, 1512, 1460, 1444, 1399, 1382, 1254,1224, 1187, 1148, 1063, 1036, 939, 827, 735. Purity: 96.4% (Method A).

4-(4-(4-(4-(((2R,4R)-2-(2,4-dichlorophenyl)-2-methyl-1,3-dioxolan-4-yl)methoxy)phenyl)piperazin-1-yl)phenyl)-2-propyl-2,4-dihydro-3H-1,2,4-triazol-3-one(22a). ¹H NMR (500 MHz, CDCl₃) δ 7.61 (m, 2H), 7.41 (m, 3H), 7.23 (m,1H), 7.02 (m, 2H), 6.94 (m, 2H), 6.89 (m, 2H), 4.32 (m, 1H), 4.11 (m,1H), 4.01 (m, 1H), 3.96 (m, 1H), 3.82 (m, 3H), 3.36 (m, 4H), 3.23 (m,4H), 1.84 (m, 2H), 1.81 (s, 3H), 0.98 (t, J=7.4 Hz, 3H). ¹³C NMR (126MHz, CDCl₃) δ 153.0, 152.1, 150.6, 145.9, 138.1, 134.5, 133.8, 132.8,131.1, 128.8, 126.7, 125.8, 123.5, 118.4, 116.6, 115.4, 109.0, 73.9,69.2, 66.9, 50.6, 49.2, 47.2, 25.7, 22.0, 11.1. DART-HRMS: m/z calcd.for C₃₂H₃₆Cl₂N₅O₄ [MH]⁺, 624.2144; Found: 624.2143. IR (solid) vmax3125, 3059, 2935, 2876, 2829, 1703, 1686, 1614, 1585, 1551, 1510, 1454,1405, 1375, 1336, 1297, 1225, 1194, 1143, 1094, 1035, 944, 869, 814,733. Purity: 96.6% (Method B).

4-(4-(4-(4-(((2S,4R)-2-(2,4-dichlorophenyl)-2-methyl-1,3-dioxolan-4-yl)methoxy)phenyl)piperazin-1-yl)phenyl)-2-propyl-2,4-dihydro-3H-1,2,4-triazol-3-one(23a). ¹H NMR (500 MHz, CDCl₃) δ 7.64 (d, J=8.4 Hz, 1H), 7.60 (s, 1H),7.41 (m, 2H), 7.38 (d, J=2.1 Hz, 1H), 7.20 (m, 1H), 7.02 (d, J=8.9 Hz,2H), 6.90 (d, J=8.9 Hz, 2H), 6.73 (d, J=8.9 Hz, 2H), 4.64 (m, 1H), 4.30(m, 1H), 3.94 (m, 1H), 3.82 (t, J=7.2 Hz, 2H), 3.73 (m, 2H), 3.35 (m,4H), 3.21 (m, 4H), 1.83 (m, 2H), 1.78 (s, 3H), 0.98 (t, J=7.4 Hz, 3H).¹³C NMR (126 MHz, CDCl₃) δ 152.8, 152.1, 150.5, 145.8, 139.3, 134.3,133.8, 132.7, 130.9, 128.5, 126.6, 125.8, 123.5, 118.4, 116.6, 115.2,109.1, 99.9, 75.0, 68.3, 67.3, 50.6, 49.2, 47.2, 25.8, 22.0, 11.1. HRMS:m/z calcd. for C₃₂H₃₆Cl₂N₅O₄ [MH]⁺, 624.2144; Found: 624.2142. IR(solid) vmax 2966, 2932, 2904, 2830, 1698, 1609, 1585, 1550, 1511, 1461,1402, 1385, 1336, 1296, 1224, 1189, 1148, 1097, 1036, 940, 875, 824,735. Purity: 96.5% (Method B).

4-(4-(4-(4-(((2S,4S)-2-(2,4-dichlorophenyl)-2-methyl-1,3-dioxolan-4-yl)methoxy)phenyl)piperazin-1-yl)phenyl)-2-propyl-2,4-dihydro-3H-1,2,4-triazol-3-one(24a). ¹H NMR (500 MHz, CDCl₃) δ 7.64 (d, J=8.4 Hz, 1H), 7.60 (s, 1H),7.41 (d, J=8.8 Hz, 2H), 7.38 (d, J=2.1 Hz, 1H), 7.02 (m, 2H), 6.90 (d,J=8.5 Hz, 2H), 6.73 (m, 2H), 4.60 (m, 1H), 4.30 (m, 1H), 3.94 (m, 1H),3.81 (t, J=7.2 Hz, 2H), 3.73 (m, 2H), 3.35 (m, 4H), 3.22 (m, 4H), 1.83(m, 2H), 1.78 (s, 3H), 0.98 (t, J=7.4 Hz, 3H). ¹³C NMR (126 MHz, CDCl₃)δ 153.0, 152.1, 150.6, 145.9, 138.1, 134.5, 133.8, 132.8, 131.1, 128.8,126.7, 125.8, 123.5, 118.4, 116.6, 115.4, 109.0, 73.9, 69.2, 66.9, 50.6,49.2, 47.2, 25.7, 22.0, 11.1. DART-HRMS: m/z calcd. for C₃₂H₃₆Cl₂N₅O₄[MH]⁺, 624.2144; Found: 624.2140. IR (solid) vmax 2919, 2876, 2849,1693, 1601, 1585, 1555, 1509, 1450, 1403, 1375, 1331, 1295, 1225, 1188,1149, 1095, 1032, 1017, 943, 871, 814, 734. Purity: 95.4% (Method B).

4-(4-(4-(4-(((2R,4S)-2-(2,4-dichlorophenyl)-2-methyl-1,3-dioxolan-4-yl)methoxy)phenyl)piperazin-1-yl)phenyl)-2-propyl-2,4-dihydro-3H-1,2,4-triazol-3-one(25a). ¹H NMR (500 MHz, CDCl₃) δ 7.61 (m, 2H), 7.41 (m, 3H), 7.23 (m,1H), 7.03 (m, 2H), 6.95 (m, 2H), 6.89 (m, 2H), 4.32 (m, 1H), 4.11 (m,1H), 4.00 (m, 1H), 3.94 (m, 1H), 3.82 (m, 3H), 3.37 (m, 4H), 3.24 (m,4H), 1.83 (m, 2H), 1.81 (s, 3H), 0.98 (t, J=7.4 Hz, 3H). ¹³C NMR (126MHz, CDCl₃) δ 153.0, 152.1, 150.5, 145.9, 138.0, 134.5, 133.8, 132.7,131.1, 128.8, 126.7, 125.9, 123.5, 118.4, 116.6, 115.4, 109.0, 73.9,69.2, 66.9, 50.6, 49.1, 47.2, 25.7, 22.0, 11.0. DART-HRMS: m/z calcd.for C₃₂H₃₆Cl₂N₅O₄ [MH]⁺, 624.2144; Found: 624.2105. IR (solid) vmax2918, 2876, 2849, 1693, 1585, 1555, 1509, 1464, 1449, 1374, 1330, 1294,1224, 1187, 1149, 1095, 1035, 964, 942, 872, 817, 734. Purity: 97.8%(Method B).

Biological Assay Protocols.

General Information.

Protocols for general cell culture, qPCR and Hh inhibition in C3H10T1/2and ASZ cells are as previously described.²⁸Protocols for the initiationand growth of Math1-Cre-ER;Ptc^(fl/fl) medulloblastoma tumors, isolationand in vitro culture of MERP MB cells, as wells as theanti-proliferation and qPCR studies performed in these cells were aspreviously described.^(a) Sonic Hedgehog (C25II) recombinant mouseprotein was purchased from Life Technologies. aData was analyzed usingGraphPad Prism 5 and reported values represent mean±SEM for at least twoseparate experiments performed in triplicate.

HUVEC Cell Viability and Proliferation.

HUVEC cell proliferation was assessed by measuring cellular metabolicactivity using standard MTS/PMS protocols according to manufacturer'sinstructions. Briefly, HUVECs (3,000 cells/well) were seeded in a96-well plate and allowed to attach overnight. Cells were treated withvarying concentrations of drug as indicated and proliferation wasassessed after 72 h with MTS.

Tube Formation Assay.

Matrigel (BD Biosciences) was diluted 1:1 with DMEM (matrigel proteinconcentration no less than 3 mg/ml) and used to coat the wells of a24-well tissue culture dish (280 uL per well). Plates were incubated at37° C. for no more than 1 h until the matrigel solidified. HUVECs weresuspended in M199 media with 1% FBS and penicillin/streptomycin and50,000 cells were added to each well. Plates were incubated at 37° C.for 20-30 min to allow HUVECs to attach to the matrigel. Cells weretreated with control (DMSO), known angiogenic inhibitor suramin (10 uM),or varying doses (10, 1, 0.1 μM) of drug and incubated for 16 h. Phasecontrast images were taken from multiple locations in each well (8/well)on an inverted microscope and tube formation parameters quantified usingImage J software (NIH).

Results

A first generation series of ITZ analogues that systematically truncatesthe ITZ scaffold from both the left- and right-hand side to identify keystructural features required for inhibition of both Hh signaling andangiogenesis (Table 1a) has been prepared.

Com- pound R₁ R₂ R₃  1a, ITZ

 2a —CH₃

 3a

 4a

—CH₃

 5a —CH₃ —CH₃

 6a

 7a

 8a

 9a

10a

—NO₂ 11a

—NH₂

Evaluation of this first generation series provided keystructure-activity relationship (SAR) data that was subsequentlyutilized for the design of a second generation of stereo chemicallydefined ITZ derivatives based on analogue 2a (Table 2). The secondgeneration series was designed to determine whether a specificstereoisomer of key ITZ analogue 2a was responsible for its anti-Hhand/or anti-angiogenic properties.

TABLE 2 Second generation, stereochemically defined analogues of 2.

Compound R₁ R₂ Final Stereochemistry 12a

Trans-2,4 13a

Cis-2,4 14a

Trans-2S,4S,2′S 15a

Cis-2R,4S,2′S 16a

Trans-2R,4R,2′S 17a

Cis-2S,4R,2′S 18a

Trans-2S,4S,2′R 19a

Cis-2R,4S,2′R 20a

Trans-2R,4R,2′R 21a

Cis-2S,4R,2′R 22a

Trans-2R,4R 23a

Cis-2S,4R 24a

Trans-2S,4S 25a

Cis-2R,4S

Analogues 1a-25a were synthesized following slightly modified literatureprocedures. All of these analogues contain the phenyl-piperazine-phenyllinker region of ITZ, which is initially constructed by a coupling stepbetween commercially available N-(4-methoxyphenyl)-piperazine 26a and1-chloro-4-nitrobenzene 27a to yieldN-(4-hydroxylphenyl)-N′-(4-nitrophenyl)-piperazine 28a (Scheme 1a). Thenitro moiety in 28a is reduced to the aniline 29a in the presence ofhydrazine monohydrate and 10% palladium on charcoal. The anilineundergoes a series of well-characterized transformations to ultimatelyprovide the key triazolone intermediate 32a. The methoxy substituent instarting material 26a serves as a protecting group throughout Scheme 1and while other literature sources protect this phenol with amethoxymethyl (MOM) group, we found the synthetic scheme utilizing theMOM protection to be significantly less efficient in generating thecorresponding key intermediate.

^(a)Reagents and conditions: (a) K₂CO₃, reflux, 12 h, 82%; (b) Pd/C,NH₂NH₂—H₂O (10 eq), reflux, 3.5 h, 71%; (c) Pyr (17 eq), ClCOOPh (1.1eq), 3 h, 90%; (d) NH₂NH₂—H₂O (5.5 eq), reflux, 3 h, quant; (e)formamidine acetate (4.5 eq), acetic acid, reflux, 3 h, 91%.

Triazolone 32a was alkylated with either commercially available alkylbromides (37a-38a) or alkyl brosylates (35a-36a), which were preparedfrom the corresponding commercially available and stereochemicallydefined alcohols (33a-34a) (Scheme 2a). The methoxy group in thelinker/triazolone/side chain intermediates (39a-42a) was removed with48% hydrobromic acid in toluene to afford the phenols 43aa-46. It isimportant to note that alkylation of the triazolone results in aninversion of stereochemistry for the defined side chains; for example,(R)-brosylate 35a generates (S)-intermediates 39a and 43a.

Reagents and conditions: (a) Et₃N, BsCl (1.3 eq), RT, 3 h; 30-50%; (b)Cs₂CO₃, brosyl/bromo alkyl chain, RT, 12 h, 45-88% (c) 48% HBr, toluene,reflux, 12 h, 55-80%.

The dioxolane regions were obtained via two different mechanisms due totheir structural differences. Dioxolane regions that contained thetriazole moiety could not be directly formed under standard ketalizationprocedures due to the basicity of the triazole functionality. For theseintermediates, the triazole moiety (50a) was added to varioushalogenated ketones (47a-49a) to form the triazole containing ketones(51a-53a). In parallel, tosylated glycerol 56a was formed throughstandard tosylation of the commercially available dioxolane 54a andsubsequent acid-mediated hydrolysis (Scheme 3a). Final dioxolane regionintermediates containing the triazole moiety (57a-59a) were preparedthrough ketalization of 51-53 and 56 with triflic acid.

The des-triazole dioxolane regions (61a, 61a′ and 61a″ and 64a-67a) weresynthesized under standard ketalization reaction conditions utilizing aDean-Stark apparatus (Scheme 4a). Stereochemically defined tosylatedglycerols 62a and 63a were prepared via the method described above forthe racemic mixture and each of the protected glycerols was used toketalize 2,4-dichloroacetophenone. For the stereochemically defineddioxolanes, the cis-isomer was predominantly formed (˜3:1 cis:trans) andthe isomers were easily separable via column chromatography.

Tosylated dioxolane regions were coupled with the linker/triazolone/sidechain region phenols in anhydrous dimethyl sulfoxide with cesiumcarbonate to yield the final analogues 1a-8a and 12a-25a (Scheme 5aA).The truncated triazolone analogues (10a-11a) were synthesized in reverseorder.^(a) The unprotected linker region 68a was prepared in the samemanner as 28a in Scheme 1a (Scheme 5aB). This linker region intermediatewas then coupled with the tosylated dioxolane region 59a under similarconditions described above to afford truncated analogue 10a, which wasreduced to the aniline with palladium on charcoal (10%) in the presenceof hydrazine monohydrate to afford ITZ analogue 11a. Overallpurification methods varied for each ITZ analogue, particularly amongstthe first generation series that contained the triazole moiety.Purification methods for all compounds are listed in the experimentalinformation.

Biological Evaluation:

The initial evaluation of first generation ITZ analogues (la-11a) as Hhpathway inhibitors was performed by monitoring endogenous Gli1 mRNAlevels in C3H10T1/2 cells, an Hh-dependent mouse embryonic fibroblast(MEF) at a single concentration (1 μM). In this well-studied modelsystem for evaluating small molecule inhibition of Hh signaling,addition of an exogenous Hh agonist (recombinant Hh ligand or smallmolecule) results in a characteristic increase in Gli1 mRNA expression.Concomitant incubation with a pathway inhibitor reduces Gli1 expression.Analogues that reduced Gli1 mRNA levels below 20% in this assay weresubsequently evaluated for their ability to reduce Gli1 mRNA expressionlevels in a concentration-dependent fashion in these cells and theHh-dependent murine BCC cell line ASZ. In addition, several compoundswere evaluated for their anti-proliferative effects in primaryHh-dependent medulloblastoma cells isolated from conditional patchedknockout (Math1-Cre-ER;Ptc^(fl/fl), MERP) mice. Based on the primarynature of the MERP cells, only a small subset of 1^(st) and 2^(nd)generation ITZ analogues were chosen for evaluation in these cells.Finally, each of these analogues was evaluated for its ability toinhibit proliferation in human umbilical vein epithelial cells (HUVECs).In vivo angiogenesis is dependent on endothelial cell proliferation;therefore, the inhibition of HUVEC proliferation is commonly utilized asan early stage in vitro model of anti-angiogenic activity. In addition,the identification of ITZ as an anti-angiogenic compound was establishedvia this assay. The results for these assays are given in Table 3a.

Evaluation of the first generation of ITZ analogues indicated severalstructural features that appear necessary for Hh pathway inhibition.While the absolute stereochemistry at the 2′-position of the sec-butylside chain does not appear important, 6a and 7a are equipotent in theMEF and ASZ cell lines, removing the methyl moiety (8a) significantlyreduces the overall activity of the scaffold. Interestingly, removingthe side chain (9a) or the triazolone/side chain (10a-11a) did notaffect the Hh inhibitory activity of the scaffold as each of theseanalogues also demonstrated potent inhibition of pathway signaling ineach cell line with IC₅₀ levels comparable to ITZ. In regards tomodifications to the dioxolane region, removal of the chlorine atoms onthe phenyl ring had minimal effects (3a), while complete truncation ofthe phenyl ring (4a) resulted in a significant loss of anti-Hh activity.Removal of the triazole moiety (2a) had no effect on the ability of thescaffold to inhibit Hh signaling. Not surprisingly, removal of both thetriazole and phenyl ring (5) or complete truncation to the phenol (45a),significantly reduced Hh inhibitory activity. Our synthesized ITZ (1a)demonstrated comparable activity to the ITZ purchased commercially ineach of the cell lines evaluated. Finally, while each of the analoguesevaluated was equipotent in its ability to down-regulate Gli1 mRNAexpression in both the C3H10T1/2 and ASZ cells (Table 3), the ability ofITZ, 1a, and 11a to inhibit the proliferation of Hh-dependent murine MBcells was slightly reduced.

TABLE 3a In vitro activity of first generation ITZ analogues. % Gli GI₅₀(μM)^(c) expression IC₅₀ (μM)^(c) GI₅₀ (μM)^(c) MERP Compound (1 μM)^(a)C3H10T1/2^(a) ASZ^(d) HUVEC MB^(d) ITZ — 0.074± 0.14 ± 0.02 0.40 ± 0.030.44 ± 0.08 1a   1.7 ± 0.3^(b) 0.063± 0.17 ± 0.01 0.49 ± 0.09 0.6 ± 0.12a 17.8 ± 0.5 0.14 ± 0.04 0.17 ± 0.04 23.8 ± 6.7  ND 3a 13.1 ± 1.2 0.42± 0.2  0.45 ± 0.06 8.3 ± 0.7 ND 4a 61.4 ± 3.5 ND ND 18.4 ± 4.5  ND 5a71.4 ± 5.5 ND ND >100 ND 6a  6.9 ± 3.1 0.16 ± 0.04  0.14 ± 0.008 2.5 ±0.3 ND 7a  3.0 ± 0.9 0.14 ± 0.04 0.16 ± 0.07 1.7 ± 0.4 ND 8a 58.5 ± 6.4ND ND 4.7 ± 0.3 ND 9a  1.1 ± 0.2 0.043± 0.12 ± 0.03 5.8 ± 0.8 ND 10a  1.5 ± 0.05 0.13 ± 0.03 0.09 ± 0.01 8.4 ± 0.7 ND 11a   6.1 ± 1.6 0.16 ±0.06 0.12 ± 0.05 42.7 ± 4.4  0.9 ± 0.7 45a  45.7 ± 3.0 ND ND >100 ND^(a)All analogues evaluated following 24 hr incubation. ^(b)Valuesrepresent % Gli1 expression relative to recombinant Hh ligand control(set as 100%). ^(c)IC₅₀ and GI₅₀ values represent the Mean ± SEM of atleast two separate experiments performed in triplicate. ^(d)Allanalogues evaluated following 48 hr incubation.

In addition to exploring Hh pathway inhibition, this first generation ofITZ analogues was evaluated for their ability to inhibit VEGF-inducedproliferation in HUVECs, an initial step towards determining theirability to inhibit angiogenesis. The ITZ synthesized in the lab (1)demonstrated anti-proliferative activity comparable to the commerciallypurchased ITZ (GI₅₀ values=0.49 and 0.40 μM, respectively). Theremaining ITZ analogues were significantly less active than ITZ in thisassay. Several analogues were moderately active (GI₅₀ values, 1.7-8.4μM), but none were comparable to ITZ. Finally, we compared ITZ and 2afor their ability to inhibit CYP3A4. Not surprisingly, removal of thetriazole moiety completely abolished the ability of 2a to inhibit CYP3A4(IC₅₀ values=50.4 nM and >10 μM, respectively, FIG. 6).

As noted above, the synthetic route primarily utilized to access ITZresults in a 1:1:1:1 ratio of four stereoisomers that share the cisconfiguration for the triazole and ether linker around the dioxolanering. As a means to optimize our time and efforts related to thesynthesis and evaluation of our first generation series of ITZanalogues, we did not fully characterize the ratio of stereoisomerspresent in each of the analogues. Instead they were evaluated as thestereoisomeric mixtures produced via the synthetic route(s) described.In order to more fully probe the absolute structural requirements of thescaffold for potent inhibition of Hh signaling and angiogenesis, wesynthesized and evaluated a second generation series of stereochemicallydefined ITZ analogues based on the des-triazole ITZ analogue 2a. It isimportant to note that the nomenclature of the stereochemistry regardingthe dioxolane region differs between ITZ and des-triazole ITZ analogues.The triazole moiety receives priority within the fully intact ITZdioxolane region; therefore, the cis-orientation is in reference to thetriazole and ether linkage (Chart 1). By contrast, removal of thetriazole shifts priority to the phenyl ring and a cis-des-triazoleanalogue has the opposite absolute configuration between the phenyl ringand ether linker as ITZ. In addition, coupling of the dioxolane regionintermediates to the linker/side chain phenol results in the oppositenomenclature assignment for the final ITZ analogue, for example, finalanalogues that have the 4R stereochemistry were prepared from the (S)tosylate 62a.

Evaluation of our stereochemically defined analogues for their abilityto inhibit Hh signaling in the C3H10T1/2 and ASZ cell lines providedboth interesting and confounding results. Overall, the majority of thecompounds evaluated (12a-25a, Table 4) were more active in the ASZcells, with numerous analogues exhibiting 100- to 1000-fold improvementin activity in these cells when compared to the MEFs. Several analoguesthat were inactive in the C3H10T1/2 MEFs at concentrations up to 10 μM(15a, 24a-25a) induced potent down-regulation of Gli1 (IC₅₀ values=0.55,0.2, and 0.54 μM, respectively). In addition, several other compoundswith modest inhibitory effects in the MEF cell line (13a, 17a, and 21a)exhibited low nanomolar IC₅₀ values in the ASZ cells.

Even with the conflicting results between cell lines, severalinteresting SAR developments with respect to optimal configuration wereidentified for our second generation ITZ analogues. First, these resultsreiterated that the orientation of the methyl moiety in the sec-butylside chain is less important, as long as it is present (Table 4a).Compounds that included a propyl moiety were significantly less activethan analogues maintaining the same orientation around the dioxolanering (i.e. 17a and 23a; 18a and 24a) and incorporating the sec-butylmoiety. ITZ analogues with the trans-orientation around the dioxolanegenerally demonstrated enhanced down-regulation of Gli1 mRNA expressionin both cell lines when compared to the corresponding cis analogues.Finally, compounds containing the 4R configuration were generally moreactive than corresponding analogues with the 4S configuration anddes-triazole analogues with the trans 2R,4R configuration (16a, 20a, and22a) demonstrated the most comparable activity between the two celllines.

TABLE 4a In vitro activity of second generation ITZ analogues. GI₅₀(μM)^(a) Com- IC₅₀ (μM)^(a) GI₅₀ (μM)^(a) MERP pound C3H10T1/2^(b)ASZ^(c) HUVEC MB^(c) 12a 0.091 ± 0.02  0.077 ± 0.01  3.32 ± 0.55 2.0 ±1.3 13a 0.85 ± 0.1  0.071 ± 0.02  3.69 ± 1.1  2.9 ± 1.9 14a  1.1 ± 0.172.5 ± 0.7 7.7 ± 4.4 ND 15a >10 0.55 ± 0.07 78.3 ± 17.3 ND 16a  0.47 ±0.001 0.38 ± 0.07 18.3 ± 11.8 ND 17a 1.85 ± 0.09  0.15 ± 0.038 2.5 ± 0.70.39 ± 0.2  18a 0.54 ± 0.24 0.13 ± 0.04 12.1 ± 4.7  ND 19a 6.6 ± 0.3 2.8± 0.9 65.0 ± 11.5 ND 20a 0.19 ± 0.04 0.022 ± 0.01  53.2 ± 25.2 0.6 ± 0.221a 2.4 ± 0.6 0.024 ± 0.02  12.8 ± 2.9  1.0 ± 0.5 22a 4.1 ± 1.5  1.3 ±0.65 26.5 ± 3.5  ND 23a >10 1.4 ± 0.5 49.9 ± 15.9 ND 24a >10  0.2 ± 0.0884.1 ± 33.5 22.4 ± 12   25a >10 0.54 ± 0.06 24.9 ± 2.2  ND PSZ 0.14 ±0.02 0.54 ± 0.05  1.6 ± 0.02 1.5 ± 0.3 ^(a)IC₅₀ and GI₅₀ valuesrepresent the Mean ± SEM of at least two separate experiments performedin triplicate. ^(b)All analogues evaluated following 24 hr incubation.^(c)All analogues evaluated following 48 hr incubation.

Based on the anti-Hh activity of the stereochemically defined compoundsin the C3H10T1/2 and ASZ cells, we evaluated several of these ITZanalogues in the primary MB cell line. The specific analogues selectedfor the anti-proliferative studies were chosen either because they werepotent in both cell lines (12a and 20a) or because they weresignificantly less active in the MEFs (13a, 17a, 21a, 24a). Measuringthe anti-proliferative effects of these analogues in the Hh-dependentprimary cell culture would not only provide additional in vitro data forthe most active trans analogues, but might also serve to clarify thediscrepancies in anti-Hh activity between the two immortalized celllines. Not surprisingly, the mixtures of trans-(12a) and cis-(13a)stereoisomers were less active than the single stereochemically definedanalogues. The most active analogue in this assay was 17a (GI₅₀=0.39μM), which has the cis 2R,4R configuration around the dioxolane moiety.Finally, the reduced anti-proliferative activity demonstrated foranalogue 24a further highlights the importance of the methyl group onthe side chain for Hh inhibition. Based on the anti-proliferativeresults in the MB cells, we chose to evaluate ITZ and three analogues(17a, 20a, and 21a) for their ability to down-regulate endogenous Gli1mRNA expression in the MERP MB cell line. Along with ITZ, each of thesecompounds exhibited potent down-regulation of mRNA expression in thisassay further demonstrating their ability to inhibit Hh signaling (Table5a). It is important to note that the anti-Hh activity of the ITZanalogues in the MERP MB cells more closely correlated with the dataobtained in the ASZ cells, suggesting that the immortalized BCC cellline may be a more appropriate early stage in vitro cellular model of Hhsignaling for evaluating analogues based on the ITZ scaffold.

TABLE 5a Down-regulation of Gli1 mRNA in MERP MB cells. Compound^(a)IC₅₀ (μM)^(b) ITZ 0.39 ± 0.06 17a 0.26 ± 0.12 20a 0.19 ± 0.07 21a 0.29 ±0.08 ^(a)All analogues evaluated following 48 hr incubation. ^(b)IC₅₀values represent the Mean ± SEM of two separate experiments.

Following the initial identification of ITZ as an Hh pathway inhibitor,several additional azole anti-fungals were evaluated for their anti-Hhproperties. Interestingly, the closely related, stereochemically definedtriazole anti-fungal posaconazole was not explored in these earlystudies. Based on our findings that ITZ analogue 20a was the mostconsistent in its ability to inhibit pathway activity across multipleHh-dependent cell lines, we also evaluated PSZ, which shares the sameorientation around its tetrahydrofuran (THF) ring, in each of theseassays. PSZ demonstrated potent down-regulation of Gli1 expression inthe MEF and ASZ cell lines; however, it was significantly less active inthe MERP cells. Taken together, these data strongly suggesting that thedioxolane and hydrophobic side chain of ITZ are more active than thecorresponding THF and hydroxylated side chains of PSZ.

The second generation of ITZ analogues was also evaluated in the HUVECsfor their anti-angiogenic activity. In a fashion similar to our initialseries, each analogue evaluated in this assay was significantly lessactive than the commercially purchased ITZ. Interestingly, thetrans-(12a) and cis-(13a) dioxolane mixtures were both more potent thanthe complete mixture (2a). Our results did not yield a clear SAR patternthat distinguished one stereo-defined dioxolane region to be more potentagainst HUVEC anti-proliferation he stereoisomer that had the lowestIC₅₀, 17a, contains the (R)-cis-dioxolane region and the (S)-sec-butylgroup (IC₅₀=2.5±0.7 μM). When only the stereochemistry of the sec-butylmoiety is inverted (21a), activity is attenuated (IC₅₀=12.8±2.9 μM).Overall, the (S)-sec-butyl analogues have lower IC₅₀ values than thecorresponding (R)-sec-butyl analogues. The same trend held true for the(R)-trans-dioxolane analogues (16a and 20a); this was the most potentorientation determined from the Hh pathway evaluation. These resultsdiffer from the Hh pathway inhibitory activity of the scaffold in thatthe stereochemistry of the side chain region did not play a significantrole in achieving potent inhibition. Taken together, there is not onedioxolane region that can be readily determined as the most potentacross the second generation of ITZ analogues for inhibitingproliferation of HUVEC cells.

Based on the lack of clear SAR in the HUVEC cells, we sought to furtherexplore the anti-angiogenic properties of ITZ and our analogues byevaluating their ability to inhibit tube formation in HUVECs grown onMatrigel. Under normal conditions, HUVECs plated and grown on Matrigelmigrate towards each other, align, and form tubes that resemble in vivocapillary beds. This assay is generally considered a more robust modelof angiogenesis as it requires several aspects of proper vesselformation, including adhesion, migration, and tube formation. As tubeformation assays are inherently lower-throughput, we chose to evaluateonly a few select compounds, including, ITZ, 2a, 18a, 21a, and thewell-characterized angiogenesis inhibitor suramin (positive control).These analogues were chosen for evaluation based on both their activityin the anti-proliferation assays and their overall structure in anattempt to provide the most relevant preliminary information withrespect to the ability of this scaffold to inhibit angiogenesis.

Inhibition of tube formation (as measured by overall tube length andtotal tube junctions) by ITZ and 2a were comparable to that of thepositive control suramin at 10 μM; however, neither of thestereochemically defined analogues (18a and 21a) were active at thisconcentration (FIGS. 12 and 13). ITZ also demonstrated significantinhibition of tube formation at 1 μM. A decrease in activity wasobserved at 1 μM for analogue 2a, highlighting the importance of thetriazole moiety for optimal anti-angiogenic activity of the scaffold.Interestingly, there was minimal correlation between the ability of acompound to inhibit anti-proliferation and tube formation in the HUVECcells. ITZ was most active in both assay systems; however, analogues 18aand 21a were two-fold more active in the anti-proliferation assay than2a, yet they were essentially inactive in the tube formation assay.Taken together, these data suggest that the triazole of ITZ is importantfor the anti-angiogenic activity of the scaffold.

Discussion and Conclusions:

Significant modifications to multiple regions of the ITZ scaffoldresulted in compounds that retained the ability to inhibit Hh signalingin Hh-dependent MEFs, murine BCC cells, and murine MB cells. Bycontrast, every analogue evaluated was significantly less active thanITZ in the anti-angiogenic assays performed in HUVECs. Preliminary dataprovides strong evidence that ITZ inhibits the Hh pathway throughSmoothened (Smo), a key regulator of this signaling cascade. As neitherSmo nor Hh signaling are known to play a role in angiogenesis, it isreasonable to hypothesize that the anti-angiogenic properties of ITZ aremediated through a distinct, as yet unidentified, cellular target notassociated with this developmental pathway; however, further studies areneeded to definitively characterize Smo/ITZ binding interactions andidentify the cellular protein(s) responsible for its ability to inhibitcellular angiogenesis.

A key goal for our initial ITZ analogue series was to determine whetherthe triazole moiety was essential for the anti-cancer properties of thescaffold. Removal of the triazole from the dioxolane region (2a) had noeffect on the ability to inhibit Hh signaling in either cell line;however, it did significantly affect the anti-angiogenic properties ofthe scaffold. This also provides further evidence that the anti-canceractivities of ITZ are mediated through distinct cellular mechanisms andhighlights the importance of the triazole for the ability of ITZ toinhibit angiogenesis. Not surprisingly, removal of the triazolecompletely abolished inhibition of CYP3A4, the main detrimental sideeffect of ITZ, providing an improved lead scaffold for furtherdevelopment as an inhibitor of Hh signaling.

After establishing that the triazole functionality is not required forHh inhibition, we prepared and evaluated two additional series ofanalogues to (1) determine whether truncation at other locationsaffected the anti-Hh properties of the scaffold and (2) identify theoptimal stereochemical orientation of our new lead 2. The truncatedanalogues were not only designed to identify the pharmacophore for ITZinhibition of Hh signaling, but also to reduce the overall size of ITZ(molecular weight=705.6 g/mol) to improve both its drug-like propertiesand the overall synthetic efficiency of preparing these compounds.Overall, the dioxolane region was not amenable to further truncation;however, removing the triazolone/side chain region did not affect theability of the scaffold to inhibit Hh signaling in any of theHh-dependent cell lines (analogue 11a). For the stereochemically definedanalogues of 2a, the trans orientation was preferred for the dioxolaneregion and more specifically the trans 2R,4R analogues were consistentlymore active than other analogues with their respective side chainorientation in the immortalized cell lines. In the primary murine MBcells, a cis analogue (17a) with the 4R orientation also demonstratedpotent anti-proliferative effects, suggesting that the (R)-configurationat the 4-position of the dioxolane ring might be more important for Hhinhibition than the overall cis- or trans-orientation. This is furtherhighlighted by the significant reduction in anti-proliferative activityfor analogue 24a (trans-2S,4S).

While the active first generation ITZ analogues demonstrated comparableHh inhibition in both the C3H10T1/2 and ASZ cell lines, the secondgeneration, stereochemically defined analogues were generally moreactive in the ASZs. Several possibilities exist to explain thedifferential activity identified for the second generation analogues.First, it is possible that decreased permeability for thestereochemically defined compounds in the C3H10T1/2 cells prevents theirintracellular accumulation at concentrations required for potentactivity. Second, the cellular target that mediates the anti-Hh activityof the ITZ scaffold (presumably Smo) may be more responsive to thestereochemically defined analogues in the ASZ cells. In addition, Hhsignaling in the C3H10T1/2 cells must be activated through the additionof an exogenous agonist (for these assays, recombinant sonic Hh ligand),while Hh signaling and Gli1 overexpression are constitutively active inthe ASZ cells due to a heterozygous mutation in the PTCH1 allele.Up-regulation of pathway signaling with the Hh ligand in the MEFs mayresult in a level of Gli1 overexpression that cannot be fully overcomeby the ITZ analogue(s). Finally, complete inhibition of Hh signaling forthe ITZ scaffold may rely on the presence of multiple stereoisomers tofully inhibit its target, which could explain why the isomeric mixturesin the first generation demonstrated comparable activity across bothcell lines. These discrepancies are currently being addressed in ongoingstudies to more fully understand the mechanisms that govern ITZ-mediatedinhibition of both cell lines.

A consideration for the further development of these and other ITZanalogues as potential Smo antagonists is that multiple forms of mutantSmo have been identified in both BCC and MB patients receiving a smallmolecule Smo antagonists. These mutations in Smo oftentimes renderpatients insensitive to further treatment with the Smo antagonists thathave been approved by the FDA. A key rationale for developing ITZanalogues as Hh pathway inhibitors is that ITZ has previouslydemonstrated the ability to inhibit several resistant forms of Smo invitro and in vivo, however, any further development of these analoguesas Hh inhibitors must demonstrate that they also prevent pathwaysignaling in the presence of mutant Smo. Attempts to circumvent mutantSmo by Developing small molecule Hh pathway inhibitors that functiondownstream of Smo at the level of the Gli transcription factors hasemerged as a potential strategy to circumvent mutant Smo; however, allof the Gli inhibitors reported to date demonstrate only modestinhibition of Hh signaling, suggesting more studies are necessary todetermine whether directly targeting Gli(s) is a valid therapeuticstrategy.

In conclusion, we have synthesized and evaluated two series of ITZanalogues for their ability to inhibit both Hh signaling andangiogenesis.

Certain compounds have duplicate numbers as indicated below.

Name 1 Name 2 ITZ itraconazole 1, ITZ (Table 1a) Analogue 1 and 95 2a(Table 1a) 93 3a (Table 1a) 94 4a (Table 1a) 96 5a (Table 1a) 27-3014a-17a (Table 2a) 23-26 18a-21a (Table 2a) 31-34 22a-25a (Table 2a)

The use of the terms “a” and “an” and “the” and similar referents(especially in the context of the following claims) are to be construedto cover both the singular and the plural, unless otherwise indicatedherein or clearly contradicted by context. The terms first, second etc.as used herein are not meant to denote any particular ordering, butsimply for convenience to denote a plurality of, for example, layers.The terms “comprising”, “having”, “including”, and “containing” are tobe construed as open-ended terms (i.e., meaning “including, but notlimited to”) unless otherwise noted. Recitation of ranges of values aremerely intended to serve as a shorthand method of referring individuallyto each separate value falling within the range, unless otherwiseindicated herein, and each separate value is incorporated into thespecification as if it were individually recited herein. The endpointsof all ranges are included within the range and independentlycombinable. All methods described herein can be performed in a suitableorder unless otherwise indicated herein or otherwise clearlycontradicted by context. The use of any and all examples, or exemplarylanguage (e.g., “such as”), is intended merely to better illustrate theinvention and does not pose a limitation on the scope of the inventionunless otherwise claimed. No language in the specification should beconstrued as indicating any non-claimed element as essential to thepractice of the invention as used herein.

While the invention has been described with reference to an exemplaryembodiment, it will be understood by those skilled in the art thatvarious changes may be made and equivalents may be substituted forelements thereof without departing from the scope of the invention. Inaddition, many modifications may be made to adapt a particular situationor material to the teachings of the invention without departing from theessential scope thereof. Therefore, it is intended that the inventionnot be limited to the particular embodiment disclosed as the best modecontemplated for carrying out this invention, but that the inventionwill include all embodiments falling within the scope of the appendedclaims. Any combination of the above-described elements in all possiblevariations thereof is encompassed by the invention unless otherwiseindicated herein or otherwise clearly contradicted by context.

1. A method of treating cancer in a subject in need thereof, comprisingadministering a therapeutically effective amount of the compound ofFormula (I)

wherein Q is O or CH₂; each Ar is independently unsubstituted orsubstituted aryl; J is O or S; R¹ is C₁₋₆ alkyl optionally substitutedwith an amino, a C₁₋₆ alkylamino, a C₁₋₆ dialkylamino, an N-acylamino,—COOH, an aryl, a heterocycloalkyl, pyrrolidine, pyrrole, or pyridinylgroup; R² is C₁₋₆ alkyl or unsubstituted or substituted aryl orheteroaryl; R³ is H or unsubstituted or substituted C₁₋₆ alkyl; R⁴ is Hor unsubstituted or substituted C₁₋₆ alkyl; or R³ and R⁴ join to form anunsubstituted or substituted 5- or 6-membered ring with the —N—(=J)-N—moiety where R³ and R⁴ form a unsubstituted or substituted C₂₋₃carbohydryl group or a unsubstituted or substituted C₁₋₂ carbohydrylgroup linked via a nitrogen to a nitrogen of the —N—(=J)-N— moiety; R⁵is H, substituted or unsubstituted C₁₋₆ alkyl, C₁₋₆ alkoxy, C₂₋₆alkanoyl, C₁₋₆ alkoxcarbonyl, C₁₋₆ haloalkyl, wherein the substitutedC₁₋₆ alkyl is substituted with 1, 2, or 3 substituents, each substituentis independently C₁₋₆ alkyl, —OH, —COOH, cyano, nitro, C₁₋₆monoalkylamine, C₁₋₆ dialkylamine, C₁₋₆ haloalkyl, C₁₋₆ haloalkoxy; apharmaceutically acceptable salt, a stereoisomeric form thereof, or acombination thereof.
 2. The method of claim 1, wherein in Formula (I) Qis O; each Ar is phenyl, pyridine, pyrazine, or pyridazine; and J is O.3. The method of claim 1, wherein in Formula (I) each Ar is phenyl. 4.The method of claim 1, wherein in Formula (I) R¹ is methyl optionallysubstituted with 1-pyrrole, 3-pyridine, 4-pyridine, phenyl,m-aminophenyl, p-aminophenyl, acetylamine, 1-pyrrolidine, amino, ordimethylamino; and R² is unsubstituted or substituted phenyl.
 5. Themethod of claim 1, wherein in Formula (I) R¹ is methyl and R² is2,4-dichlorophenyl or 2,4-difluorophenyl.
 6. The method of claim 1,wherein in Formula (I) R³ and R⁴ join to form an unsubstituted orsubstituted 5- or 6-membered ring with the —N—(=J)-N— moiety where R³and R⁴ form a unsubstituted or substituted C₂₋₃ carbohydryl group or aunsubstituted or substituted C₁₋₂ carbohydryl group linked via anitrogen to a nitrogen of the —N—(=J)-N— moiety.
 7. The method of claim1, wherein in Formula (I) R⁵ is propyl; 2′-sec-butyl, the R isomer, theS isomer, a racemate or any enantiomerically enriched form;2-hydroxypentan-3-yl, the 2R,3R-isomer, the 2S,3S, isomer, the 2R,3S,isomer, the 2S,3R isomer, or any diastereomerically enriched form;2-hydroxyprop-2-yl; or 2-hydroxyprop-1-yl, the R isomer, the S isomer, aracemate, or any enantiomerically enriched form.
 8. The method of claim1, wherein the compound has Formula (Ia)

wherein each one of X¹, X², Y¹, Y², Z¹, and Z² independently is CH,CCH₃, or N.
 9. The method of claim 8 wherein in Formula (Ia) X¹ is N andY¹, Z¹, X², Y², and Z² are CH; Y¹ is N and X¹, Z¹, X², Y², and Z² areCH; X¹ and Y¹ are N and Z¹, X², Y², and Z² are CH; X¹ and Z¹ are N andY¹, X², Y², and Z² are CH; X² is N and X¹, Y¹, Z¹, Y², and Z² are CH; Y²is N and X¹, Y¹, Z¹, X², and Z² are CH; X² and Y² are N and X¹, Y¹, Z¹,and Z² are CH; or X² and Z² are N and X¹, Y¹, Z¹, and Y² are CH.
 10. Themethod of claim 1, wherein the compound has Formula (Ib)

wherein R⁶ and R⁷ are each independently H, halo, C₁₋₆ alkyl, C₁₋₆alkoxy, C₂₋₆ alkanoyl, C₁₋₆ alkoxcarbonyl, —NH₂, —OH, —COOH, cyano,nitro, C₁₋₆ monoalkylamine, C₁₋₆ dialkylamine, C₁₋₆ haloalkyl, or C₁₋₆haloalkoxy.
 11. The method of claim 10, wherein R⁶ and R⁷ are eachindependently Cl or F.
 12. The method of claim 1, wherein the compoundis


13. The method of claim 1, wherein the cancer is dependent upon the Hhsignaling pathway.
 14. The method of claim 13, wherein the cancer isbasal cell carcinoma (BCC) or medulloblastoma (MB).
 15. The method ofclaim 13, wherein the cancer is resistant to Vismodegib.
 16. The methodof claim 1, wherein the cancer is chronic myeloid leukemia, lung cancer,prostate cancer, pancreatic cancer or bone cancer.